AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones.

The protein phosphatase encoded by bacteriophage lambda (lambda PP) belongs to a family of Ser/Thr phosphatases (Ser/Thr PPases) that includes the eukaryotic protein phosphatases 1 (PP1), 2A (PP2A), and 2B (calcineurin). These Ser/Thr PPases and the related purple acid phosphatases (PAPs) contain a conserved phosphoesterase sequence motif that binds a dinuclear metal center. The mechanisms of phosphoester hydrolysis by these enzymes are beginning to be unraveled. To utilize lambda PP more effectively as a model for probing the catalytic mechanism of the Ser/Thr PPases, we have determined its crystal structure to 2.15 A resolution. The overall fold resembles that of PP1 and calcineurin, including a conserved beta alpha beta alpha beta structure that comprises the phosphoesterase motif. Substrates and inhibitors probably bind in a narrow surface groove that houses the active site dinuclear Mn(II) center. The arrangement of metal ligands is similar to that in PP1, calcineurin, and PAP, and a bound sulfate ion is present in two novel coordination modes. In two of the three molecules in the crystallographic asymmetric unit, sulfate is coordinated to Mn2 in a monodentate, terminal fashion, and the two Mn(II) ions are bridged by a solvent molecule. Two additional solvent molecules are coordinated to Mn1. In the third molecule, the sulfate ion is triply coordinated to the metal center with one oxygen coordinated to both Mn(II) ions, one oxygen coordinated to Mn1, and one oxygen coordinated to Mn2. The sulfate in this coordination mode displaces the bridging ligand and one of the terminal solvent ligands. In both sulfate coordination modes, the sulfate ion is stabilized by hydrogen bonding interactions with conserved arginine residues, Arg 53 and Arg 162. The two different active site structures provide models for intermediates in phosphoester hydrolysis and suggest specific mechanistic roles for conserved residues.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

The R2 subunit of Escherichia coli (aerobic) ribonucleotide reductase activates molecular oxygen at its diiron center to produce a functionally essential stable tyrosyl radical from residue Y122. It was previously shown that the D84E site-directed mutant of R2 (R2-D84E) accumulates a µ-1,2-peroxodiiron(III) intermediate on the pathway to tyrosyl radical formation. This intermediate does not accumulate in the reaction of wildtype (wt) R2, but an analogous complex does accumulate during oxygen activation by the structurally similar diiron protein, methane monooxygenase hydroxylase (MMOH). Herein we describe the crystallographically determined three-dimensional structures of the reduced, diiron(II) reactant and oxidized, diiron(III) product forms of R2-D84E. The reduced R2-D84E structure differs from that of reduced wt R2 in the conformations of three carboxylate ligands, E84, E204, and E238. The adjustments in these ligands render the coordination sphere of the diiron(II) center very similar to that in reduced MMOH. In addition, a water molecule not observed in reduced wt R2 is coordinated to Fe2 in reduced R2-D84E. The oxidized R2-D84E structure is similar to that of oxidized wt R2 except in the coordination mode of E84. In R2-D84E, E84 coordinates to Fe1 in a monodentate, terminal mode and is hydrogen bonded to a water molecule also coordinated to Fe1. In wt R2, D84 is a bidentate, chelating ligand. In both R2-D84E structures, Y122 is shifted away from Fe1 such that a hydrogen bonding interaction with E84 is not possible. The observed structural adjustments suggest possible rationales for the stability of the µ-1,2-peroxodiiron(III) complex in R2-D84E. In addition, the structures expand the experimental foundation for computational investigations aimed at defining the detailed mechanistic pathways for O 2 activation at diiron(II) centers.

The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.

Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals. The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-Å resolution and a final crystallographic R-factor of 0.204. The structure reveals that the overall fold is formed by two connected seven-bladed ␤-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general. These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data. The surfaces of the ␤-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open "clamshell." The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one ␤-propeller and at the interface of the two ␤-propellers. These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p and that the orientation of the ␤-propellers with respect to one another plays a role in binding an actin-cofilin complex. In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult.The actin cytoskeleton plays a key role in cell motility, cell morphology, cytokinesis, and the organization of organelles within the cytosol. The organization of actin filaments within the cytoskeleton is tightly regulated by a number of actinbinding proteins. Among these is cofilin, an essential protein conserved in all of the eukaryotes. Cofilin and other members of the actin-depolymerizing factor/cofilin family of proteins are known to aid the rearrangement of the cytoskeleton and increase actin filament turnover by binding and severing F-actin

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The R2 subunits of class I ribonucleotide reductases (RNRs) house a diferric-tyrosyl radical (Y⅐) cofactor essential for DNA synthesis. In yeast, there are two R2 proteins, Y2 and Y4. Although both Y2 and Y4 are homologous to R2s from other organisms, Y4 lacks three conserved iron-binding residues, and its exact function is unclear. Y4 is required for assembly of the diferric-Y⅐ cofactor in Y2, and the two proteins can form both homodimeric and heterodimeric complexes. The Y2Y4 heterodimer was crystallized from a mixture of the two proteins, and its structure was determined to 2.8 Å resolution. Both Y2 and Y4 are completely ␣ helical and resemble the mouse and Escherichia coli R2s in overall fold. Three ␣ helices not observed in the mouse R2 structure are present at the Y2 N terminus, and one extra N-terminal helix is observed in Y4. In addition, one of the eight principal helices in both Y2 and Y4, ␣D, is shifted significantly from its position in mouse R2. The heterodimer interface is similar to the mouse R2 homodimer interface in size and interacting residues, but loop regions at the interface edges differ. A single metal ion, assigned as Zn(II), occupies the Fe2 position in the Y2 active site. Treatment of the crystals with Fe(II) results in difference electron density consistent with formation of a diiron center. No metal-binding site is observed in Y4. Instead, the residues in the active site region form a hydrogen-bonding network involving an arginine, two glutamic acids, and a water molecule. Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, an essential step in nucleotide metabolism in all organisms (1). By providing a balanced pool of monomeric precursors for DNA replication and repair, these enzymes play a crucial role in control of cell proliferation. Members of the largest class of RNRs, class I, are found in all eukaryotes, in many prokaryotes, and in several viruses. The catalytically active form of class I RNRs is proposed to be an ␣ 2 ␤ 2 tetramer (2). The homodimeric ␣ subunit, called R1, houses the active site and binding sites for allosteric effectors. The ␤ subunit, called R2, contains a diiron cluster that in its reduced state reacts with dioxygen to form a stable tyrosyl radical (Y⅐) and a diiron(III) cluster. This essential Y⅐ is proposed to generate a thiyl radical, located on a cysteine residue in the R1 active site, that initiates ribonucleotide reduction (3, 4).The most extensively characterized class I RNR system is that found in Escherichia coli. Crystal structures of both the E. coli R1 (5) and R2 (6, 7) proteins have been determined, and the mechanism of diferric-Y⅐ cofactor assembly has been probed by a variety of spectroscopic techniques (3). By contrast, less is known about the structure and mechanism of eukaryotic class I RNRs. A structure of mouse R2 is available (8), and cofactor formation has been investigated (9, 10). However, these studies have proved more difficult because the diiron center in mouse R2 i...