The heterotrimeric AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy producing pathways and inhibits energy consuming processes1. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer 2,3,4,5,6. AMPK is converted from an inactive to catalytically competent form by phosphorylation of the activation loop within the kinase domain7; AMP binding to the γ regulatory domain promotes phosphorylation by the upstream kinase8, protects the enzyme against dephosphorylation as well as causing allosteric activation9. We show here that ADP binding to just one of the two exchangeable AXP binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active AMPK displays significantly tighter binding to ADP than to Mg.ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg.ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. It shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity (Supplementary Fig. 1).

Article:Xiao, B., Jing, C., Wilson, J.R. et al. (7 more authors) (2003) Structure and catalytic mechanism of the human histone methyltransferase SET7/9. Nature, 421 (6923). pp.

No abstract

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein- dynactin interaction on the surface of membranous organelles, by associating with these complexes.

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.