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).

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.

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.

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.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) MgCl2/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/20 ,tM GTP (buffer A), disrupted in a glass homogenizer, and centrifuged at 640 x g for 15 min, and the supernatant was decanted. The residue was twice resuspended in 70 ml of buffer A and recentrifuged. The combined supernatants containing the cell membrane fragments were centrifuged at 186,000 x g for 90 min, and the pellet was resuspended in 50 ml of buffer A containing 2% octyl glucoside, homogenized, and gently shaken for 45 min. The solution was centrifuged at 142,000 x g for 90 min, and the supematant was filtered through a 0.45-,tm Minisart cellulose acetate filter (Sartorius). The solution (-0.4 mg of protein per ml) was loaded into a 10-ml MonoS column equili-