The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of GFP-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu222, demonstrates relatively fast maturation rate and bright green fluorescence (lambdaex = 480 nm, lambdaem = 505 nm). The reverse Gly222Glu single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high-resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr66 still stays in the native, non-oxidized form, with Calpha and Cbeta atoms in the sp3 hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250 - 300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr66 Calpha-Cbeta bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr220 in the observed effects. In particular, an alternative reaction pathway via Tyr220 rather than conventional wt Glu222 has been proposed for aceGFP maturation.

BubR1 is essential for the mitotic checkpoint which prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive varphi angles, and non-canonical packing of and between TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus provide novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of S. cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 A; R=19.9%, Rfree=25.1%) indicates that the inhibitor occupies both the glutamate- and the presumed cysteine-binding site, and disrupts the previously observed Mg2+ coordination in the ATP binding site. L-buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 A; R=18.1%, Rfree=23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme.

In addition to its endocytic function, the LDL receptor-related protein 1 (LRP1) also contributes to cell signaling events. In the current study, the potential of LRP1 to modulate the PDGF signaling pathway was investigated. PDGF is a key regulator of cell migration and proliferation and mediates the tyrosine phosphorylation of LRP1 within its cytoplasmic domain. In WI-38 fibroblasts, PDGF-mediated LRP1 tyrosine phosphorylation occurred at 37 oC, but not at 4 oC where endocytosis is minimized. Further, blockade of endocytosis with the dynamin inhibitor, dynasore, also prevented PDGF-mediated LRP1 tyrosine phosphorylation. Immunofluorescence studies revealed co-localization of LRP1 with the PDGFR following PDGF treatment within endosomal compartments, while surface biotinylation experiments confirmed that phosphorylated LRP1 primarily originates from intracellular compartments. Together, the data reveal association of these two receptors in endosomal compartments where they form a signaling complex. To study the contribution of LRP1 to PDGF signaling we used mouse embryonic fibroblasts genetically deficient in LRP1 and identified phenotypic changes in these cell lines in response to PDGF stimulation by performing phospho-site profiling. Of 38 phosphorylated proteins analyzed, 8 were significantly different in LRP1 deficient fibroblasts, and were restored when LRP1 was expressed back in these cells. Importantly, the results revealed that LRP1 expression is necessary for PDGF-mediated activation of ERK. Overall, the studies reveal that LRP1 associates with the PDGFR in endosomal compartments and modulates its signaling properties affecting the MAPK and Akt/PI3K pathways.

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is upregulated in epithelial tissues during inflammation. We demonstrated previously that NGAL is strongly upregulated by IL-1beta in an NF-kappaB-dependent manner but not by TNF-alpha, another potent activator of NF-kappaB. This is due to an IL-1beta-specific synthesis of the NF-kappaB-binding co-factor IkappaB-zeta, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-alpha in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4+ T helper cells, TH-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-beta or C/EBP-delta for NGAL induction by IL-17 and TNF-alpha as neither siRNAs against the two C/EBP mRNAs nor mutation of the C/EBP-sites in the NGAL promoter abolished IL-17 and TNF-alpha-induced upregulation of NGAL. NGAL-induction is governed solely by NF-kappaB and its co-factor IkappaB-zeta. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with siRNA against IkappaB-zeta and a total lack of activation of an NGAL promoter construct with a mutated NF-kappaB site. As IL-17-stimulation stabilizes the IkappaB-zeta transcript, we propose a model where TNF-alpha induces activation and binding of NF-kappaB to the promoters of both the IkappaB-zeta and the NGAL genes but induce only transcription of IkappaB-zeta. Co-stimulation with IL-17 leads to accumulation of IkappaB-zeta mRNA and IkappaB-zeta protein, which can bind to NF-kappaB on the NGAL promoter and thus induce NGAL expression.

Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (-)-7'-isothiocyanato-11-hydroxy-1',1'dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB2 receptor via the lipid bilayer (1). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. In work reported here, we test the hypothesis that the endogenous cannabinoid, sn-2-arachidonylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer. To this end, we have employed microsecond time scale all atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer. Results suggest that (1) 2-AG first partitions out of bulk lipid at the TMH6/7 interface; (2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6/7; (3) the entrance of the 2-AG head group into the CB2 binding pocket is sufficient to trigger breaking of the intracellular (IC) TMH3/TMH6 ionic lock and the movement of the TMH6 IC end away from TMH3; (4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics (MD) that a ligand can access the binding pocket of a Class A GPCR via the lipid bilayer and the first demonstration via MD of GPCR activation triggered by a ligand binding event.

A cancer/testis antigen, CAGE is widely expressed in various cancer tissues and cancer cell lines, but not in normal tissues except the testis. In the present study, ectopic-expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell transforming ability. Using stable HeLa transfectant clones with a tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulates both anchorage-dependent and -independent cell growth in vitro and promotes tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated CDKs, and subsequently accelerating the G1 to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even at a growth-arrested state, indicating a direct effect of CAGE on G1 cyclin expression. CAGE-induced expression of cyclin D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced Rb phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, siRNA-mediated knockdown of CAGE suppressed the expression of G1 cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclin D1 and E.

Human African Trypanosomiasis (HAT), caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of HAT is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Herein, we describe the use of an enzymatic high-throughput screen of 316,114 unique molecules to identify potent and selective inhibitors of ODC. This screen identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.

Rapid advances in genotyping and sequencing technology have dramatically accelerated the discovery of genes underlying human disease. Elucidating the function of such genes and understanding their role in pathogenesis, however, remains challenging. Here, we introduce a genomic strategy to functionally characterize such genes, and apply it to LRPPRC, a poorly studied gene that is mutated in Leigh Syndrome, French-Canadian type (LSFC). We utilize RNAi to engineer an allelic series of cellular models in which LRPPRC has been stably silenced to different levels of knockdown efficiency. We then combine genome-wide expression profiling with gene set enrichment analysis (GSEA) to identify cellular responses that correlate with the loss of LRPPRC. Using this strategy, we discovered a specific role for LRPPRC in the expression of all mitochondrial DNA (mtDNA)-encoded mRNAs, but not the rRNAs, providing mechanistic insights into the enzymatic defects observed in the disease. Our analysis shows that nuclear genes encoding mitochondrial proteins are not collectively affected by the loss of LRPPRC. We do observe altered expression of genes involved in hexose metabolism, prostaglandin biosynthesis, and glycosphingolipid biology that may either play an adaptive role in cell survival or contribute to the disease pathogenesis. The combination of genetic perturbation, genomic profiling and pathway analysis represents a generic strategy for understanding disease pathogenesis.

The tumor suppressor, Death-associated protein kinase (DAPK) is a Ca2+/CaM regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at S308 inhibits DAPK catalytic activity. Dephosphorylation of S308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), ABalphaC and ABdeltaC, as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at S308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasomal-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.

Huntington's disease (HD) is caused by an expansion of more than 35 -40 polyglutamine(polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD, and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy (AFM) to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length dependent manner. By time-lapse AFM, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures in HD pathogenesis.

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. FGF1 also plays an important role in inflammation, wound healing and restenosis. The biological effects of FGF1 are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and, therefore, require the release of the protein into the extracellular space. FGF-1 is exported through a non-classical release pathway involving the formation of a specific multi-protein complex. The protein constituents of this complex include FGF-1, S100A13, and the p40 form of synaptotagmin 1 (Syt1). As FGF1 plays an important role in tumor formation, it is clear that preventing the formation of the multi-protein complex would be an effective strategy to inhibit a wide range of cancers. To understand the molecular events in the FGF1 release pathway, we studied the FGF1-S100A13 tetrameric and FGF1-S100A13-C2A hexameric complex structures, which are both possible complexes to be formed during the non-classical pathway of FGF1 release.

We performed a comprehensive alanine scan of human alpha-defensin HNP1 and tested the ability of the resulting analogs to kill S. aureus, inhibit anthrax lethal factor, and bind HIV-1 gp120. By far, the most deleterious mutation for all of these functions was W26A. The activities lost by W26A-HNP1 were restored progressively by replacing W26 with non-coded, straight-chain aliphatic amino acids of increasing chain length. The hydrophobicity of residue 26 also correlated with the ability of the analogs to bind immobilized wild-type HNP1, and to undergo further self-association. Thus, the hydrophobicity of residue-26 is not only a key determinant of the direct interactions of HNP1 with target molecules, it also governs the ability of this peptide to form dimers and more complex quaternary structures at micromolar concentrations. Although all defensin peptides are cationic, their amphipathicity is at least as important as their positive charge in enabling them to participate in innate host defense.

The N-terminal part of the calcium regulated and phospholipid binding protein annexin AI contains peptide sequences with pro- and anti-inflammatory activities. We have earlier shown that a pro-inflammatory signal triggered by one of these peptides, Gln(9)-Lys(25), is mediated by FPR1, a member of the formylpeptide receptor family expressed in human neutrophils. To determine the core structure in Gln(9)-Lys(25), smaller peptides were generated and their capacity to activate neutrophils was determined. A peptide spanning from amino acid Glu(14)-Lys(25) was inactive whereas the activity was retained in the Gln(9)-Tyr(20) peptide. Removal of amino acids from the C- and N-terminus of Gln(9)-Tyr(20) revealed that the first amino acid (Gln(9)) was of outermost importance for activity. The core structure that activates the neutrophil NADPH oxidase to release superoxide anions was Gln(9)-Ala(10)-Trp(11)-Phe(12). This peptide also inhibited the activity induced by fMLF and WKYMVM. A structural model of the peptide agonist/FPR1 complex suggests that the transmembrane part of the binding pocket of the receptor binds optimally to a tetrapeptide. According to the model and the results presented, the N-terminal amino acid glutamine in Gln(9)-Phe(12) is located close to the bottom of the binding cleft, leaving for sterical reasons insufficient space to extend the peptide at the N-terminus. The addition of amino acids at the C-terminus will not affect binding. The model presented may be helpful in developing specific FPR1 ligands.

Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with K channels. We report the purification, synthesis and characterization of kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC50 = 33 nM) to Kv1.2 than kappaM-RIIIK (IC50 = 352 nM). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/kg b.w.), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK; whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50 = 680 nM) than that of kappaM-RIIIJ (IC50 = 3.15 muM). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit, rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

c-Jun NH2-terminal Kinases (JNKs) play a central role in the cellular response to a wide variety of stress signals. Following their activation, JNKs induce phosphorylation of substrates, which control proliferation, migration, survival, and differentiation. Recent studies suggest that JNKs may also play a role in cell cycle control, although the underlying mechanisms are largely unexplored. Here we show that JNK directly phosphorylates Cdc25C at Serine 168 during G2 phase of the cell cycle. Cdc25Cs phosphorylation by JNK negatively regulates its phosphatase activity and thereby Cdk1 activation, enabling a timely control of mitosis onset. Unrestrained phosphorylation by JNK, as obtained by a cell cycle-stabilized form of JNK or as seen in some human tumors, results in aberrant cell cycle progression. Additionally, UV-irradiation-induced G2/M checkpoint requires inactivation of Cdc25C by JNK phosphorylation. JNK phosphorylation of Cdc25C, as well as Cdc25A, establishes a novel link between stress signaling and unperturbed cell cycle and checkpoint pathways.

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized Cu-Zn superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx9C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity while CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vise versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but non-overlapping functions in cytochrome c oxidase biogenesis.

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/beta-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/beta-catenin signaling but the molecular mechanisms by which WT1 inhibits WNT/beta-catenin signaling are not completely understood. In this report, we identified a gene, CXXC5 which we have renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/beta-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/beta-catenin signaling in vitro and in vivo. WID is evolutionarily conserved and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, our results demonstrate that the WT1 negatively regulates WNT/beta-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis.

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe2+). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB to function either as a channel, or to possess an active Fe2+ membrane transport mechanism. Here, we present the crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue mant-GMPPNP. Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K+-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.