Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1 alpha is the oxygen-regulated subunit of HIF-1, an alpha beta heterodimeric complex. HIF-1 alpha is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1 alpha by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1 alpha. The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in numerous pathologies like cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small molecule and ATP-competitive PI3K inhibitors with a wide range of selectivities have entered clinical development. In order to understand mechanisms underlying isoform selectivity of these inhibitors, we developed a novel expression strategy that enabled us to determine the first crystal structure of the catalytic subunit of the class IA PI3K p110δ. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI3K inhibitors reveal that selectivity towards p110δ can be achieved by exploiting its conformational flexibility and the sequence diversity of active-site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110δ with greatly improved potencies.

Bacterial resistance to aminoglycoside antibiotics is almost exclusively accomplished through either phosphorylation, adenylylation, or acetylation of the antibacterial agent. The aminoglycoside kinase, APH(3')-IIIa, catalyzes the phosphorylation of a broad spectrum of aminoglycoside antibiotics. The crystal structure of this enzyme complexed with ADP was determined at 2.2 A. resolution. The three-dimensional fold of APH(3')-IIIa reveals a striking similarity to eukaryotic protein kinases despite a virtually complete lack of sequence homology. Nearly half of the APH(3')-IIIa sequence adopts a conformation identical to that seen in these kinases. Substantial differences are found in the location and conformation of residues presumably responsible for second-substrate specificity. These results indicate that APH(3') enzymes and eukaryotic-type protein kinases share a common ancestor.

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases (PI3Ks), occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid binding activities, for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110α/p85α. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85α N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110α. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85α-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110α. Phosphopeptide binding to p110β/p85α and p110δ/p85α complexes also induces their lipid binding. We present a crystal structure of WT p110α complexed with the p85α inter-SH2 domain (iSH2) and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110α consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110α and p85α mutations with signalling potential.

Background: Dynamin and BAR domain proteins are major components of clathrin-mediated endocytosis and other membrane-trafficking events.Results: Dynamin is recruited to membranes by BAR domain proteins, which in turn rely on interaction with dynamin to bind membranes.Conclusion: Membrane binding by dynamin and BAR domain proteins is cooperative and stimulates dynamin and GTP-dependent membrane scission.Significance: Our results imply synergy of dynamin and BAR domain proteins to promote GTP-dependent vesicle release.

Apoplastic extracts of cold-acclimated winter rye (Secale cereale 1. cv Musketeer) leaves were previously shown to exhibit antifreeze activity. The objectives of the present study were to identify and characterize individual antifreeze proteins present in the apoplastic extracts. The highest protein concentrations and antifreeze activity were obtained when the leaf apoplast was extracted with ascorbic acid and either CaClz or MgSO,. Seven major polypeptides were purified from these extracts by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under nonreducing conditions. l h e five larger polypeptides, of 19, 26, 32, 34, and 36 kD, exhibited significant levels of antifreeze activity, whereas the 11-and 13-kD polypeptides showed only weak activity. Five of these polypeptides migrated with higher apparent molecular masses on SDS gels after treatment with 0.1 M dithiothreitol, which indicated the presence of intramolecular disulfide bonds. The apparent reduction of the disulfide bonds did not eliminate antifreeze activity in four of the polypeptides that contained intramolecular disulfide bonds and exhibited significant levels of antifreeze activity. l h e amino acid compositions of these polypeptides were similar in that they were all relatively enriched in the residues Asp/Asn, Clu/Cln, Ser, Thr, Cly, and Ala; they all lacked His, except for the 26-kD polypeptide, and they contained up to 5% Cys residues. lhese polypeptides were examined with antisera to other cystinecontaining antifreeze proteins from fish and insects, and no common epitopes were detected. We conclude that cold-acclimated winter rye leaves produce multiple polypeptides with antifreeze activity that appear to be distinct from antifreezes produced by fish and inseds.

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.Overwintering plants survive freezing temperatures by forming ice within their tissues. In these plants, it has generally been believed that ice formation occurs spontaneously in large vessels in plant tissues and then spreads throughout the plant at a rate determined by the environmental temperature (14). However, it has been clearly demonstrated in other frost-tolerant organisms such as insects, reptiles, and mollusks that the process of ice formation is controlled by specific proteins. These organisms all produce proteins that function as ice nucleators to initiate extracellular ice formation (6,15,19). Moreover, freezing-tolerant insects also produce antifreeze proteins that modify the ice crystals that form outside the cells (6).We hypothesized that frost-tolerant plants may also produce proteins that control extracellular ice formation. We chose to conduct our experiments using winter rye (Secale cereale L.), an overwintering, herbaceous monocot that can survive temperatures below -200C (11). It has been shown by scanning EM that cold-acclimated winter rye leaves survive freezing temperatures by forming ice only in intercellular spaces (17). In the experiments reported here, we examined cold-acclimated rye leaves for the capacity to modify ice in a manner similar to that observed for antifreeze proteins purified from arctic and antarctic fish (5) and frost-tolerant insects (6).To prove that antifreeze proteins exist in plants, we must demonstrate that plant extracts possess two characteristics shown to be unique to previously purified antifreeze proteins. The first characteristic of antifreeze proteins is a distinctive, concentration-dependent effect on ice crystal morphology (5). In pure water, ice normally grows parallel to the basal plane (a axes) of the crystal lattice, with little growth perpendicular to the basal plane (the c axis), so that the ice crystals appear flat and round. Low concentrations (nM) of antifreeze proteins preferentially inhibit the growth of ice along the a axes so that the hexagonal prism faces of the crystal become evident. At higher concentrations (Mm) of antifreeze protein, the crystals grow predominantly along the c axis to form hexagonal bipyramids and needle-like crystals (5). The second unique characteristic of antifreeze proteins is a measurable degree of thermal hysteresis. Thermal hysteresis, the difference between freezing and melting temperatures, is determined by observing the effect of temperature on the g...

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.