Pleckstrin homology (PH) domains are small protein modules involved in recruitment of signaling molecules to cellular membranes, in some cases by binding specific phosphoinositides. We describe use of a convenient "dotblot" approach to screen 10 different PH domains for those that recognize particular phosphoinositides. Each PH domain bound phosphoinositides in the assay, but only two (from phospholipase C-␦ 1 and Grp1) showed clear specificity for a single species. Using soluble inositol phosphates, we show that the Grp1 PH domain (originally cloned on the basis of its phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) binding) binds specifically to D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ) (the PtdIns(3,4,5)P 3 headgroup) with K D ‫؍‬ 27.3 nM, but binds D-myo-inositol 1,3,4-trisphosphate (Ins(1,3,4)P 3 ) or D-myo-inositol 1,4,5-trisphosphate (Ins-(1,4,5)P 3 ) over 80-fold more weakly. We show that this specificity allows localization of the Grp1 PH domain to the plasma membrane of mammalian cells only when phosphatidylinositol 3-kinase (PI 3-K) is activated. The presence of three adjacent equatorial phosphate groups was critical for inositol phosphate binding by the Grp1 PH domain. By contrast, another PH domain capable of PI 3-K-dependent membrane recruitment (encoded by EST684797) does not distinguish Ins(1,3,4)P 3 from Ins-(1,3,4,5)P 3 (binding both with very high affinity), despite selecting strongly against Ins(1,4,5)P 3 . The remaining PH domains tested appear significantly less specific for particular phosphoinositides. Together with data presented in the literature, our results suggest that many PH domains bind similarly to multiple phosphoinositides (and in some cases phosphatidylserine), and are likely to be regulated in vivo by the most abundant species to which they bind. Thus, using the same simple approach to study several PH domains simultaneously, our studies suggest that highly specific phosphoinositide binding is a characteristic of relatively few cases.

SUMMARY Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region, but cannot explain the ‘high-affinity’ and ‘low-affinity’ classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors, and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin؊1 and dynamin؊2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin؊1 and dynamin؊2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.

Myeloproliferative neoplasms (MPNs) are blood cancers characterized by excessive production of mature myeloid cells, which result from the acquisition of somatic driver mutations in hematopoietic stem cells (HSCs). Epidemiologic studies indicate a substantial disease heritability that is among the highest known for cancers 1 . However, only a limited set of genetic risk loci have been identified, and the underlying biological mechanisms leading to MPN acquisition remain unexplained. Here, we conducted a large-scale genome-wide association study (3,797 cases and 1,152,977 controls) to identify 17 MPN risk loci (p < 5.0 × 10 −8 ), seven of which have not been previously reported. We find a shared genetic architecture between MPN risk and several hematopoietic traits spanning distinct lineages, an enrichment for risk variants mapping to accessible chromatin in HSCs, and associations of increased MPN risk with longer leukocyte telomere length and other clonal hematopoietic states, collectively implicating HSC function and self-renewal. Gene mapping identifies modulators of HSC biology and targeted variant-to-function assays suggest likely roles for CHEK2 and GFI1B in altering HSC function to confer disease risk. Overall, we demonstrate the power of human genetic studies to illuminate a previously unappreciated mechanism for inherited MPN risk through modulation of HSC function.

The final stages of dengue virus fusion are thought to occur when the membrane-proximal stem drives the transmembrane anchor of the viral envelope protein (E) toward the fusion loop, buried in the target cell membrane. Crystal structures of E have lacked this essential stem region. We expressed and crystallized soluble mutant forms of the dengue virus envelope protein (sE) that include portions of the juxtamembrane stem. Their structures represent late-stage fusion intermediates. The proximal part of the stem has both intra-and intermolecular interactions, so the chain "zips up" along the trimer seam. The penultimate interaction we detected involves the conserved residue F402, which has hydrophobic contacts with a conserved surface on domain II. These interactions do not require any larger-scale changes in trimer packing. The techniques for expression and crystallization of sE containing stem reported here may allow further characterization of the final stages of flavivirus fusion. The membrane-spanning envelope glycoprotein protein (E) of flaviviruses is both the principal determinant of icosahedral virion assembly and the fusion catalyst for merging viral and target cell membranes ( Fig. 1) (1, 2). The E protein folds into three domains, a membrane-proximal stem, and a transmembrane anchor (Fig. 1A). Various crystal structures have shown the arrangement of the three folded domains in both a dimeric prefusion conformation ( Fig. 1C) (3, 6) and a low-pHinduced postfusion trimer ( Fig. 1F) (4, 7). The hydrophobic fusion loops, buried at the dimer interface in the prefusion structure (3, 5, 6), cluster into a large hydrophobic surface at one end of the postfusion trimer (4,7,8). In this orientation, the fusion loops attach the virus to the target cell membrane. The membrane-proximal stem has two predicted amphipathic helices that lie half-buried in the outer leaflet of the viral membrane ( Fig. 1C) (9,10). For fusion to take place, this stem must span the length of domain II (Fig. 1F). A likely model is that it "zips up" along the gaps between the clustered domains, bringing together the transmembrane anchor and the fusion loops, inducing deformation of their associated membranes and leading to membrane merger.Several studies show the importance of the amino-terminal part of the stem and suggest that it forms contacts with domain II as the fusion-inducing transition proceeds (11-13). Efforts to visualize it directly in this conformation have failed, however, because including the stem residues in recombinant E generally leads to instability or aggregation of any secreted product. We describe here a method for producing sE that includes portions of the juxtamembrane stem and report its crystallization and structure determination. The structure shows that the N-terminal part of the stem zips up along the seam between adjacent domains II in the trimer. The rest of the stem in our constructs is disordered. The arrangement of the trimer core is the same as in previous, stemless structures. The results are consistent wi...

The West Nile Virus (WNV) envelope protein, E, promotes membrane fusion during viral cell entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought evidence for potential intermediates during the conformational transition by following hemifusion of WNV virus-like particles (VLPs) in a single particle format. We have introduced specific mutations into E, to relate their influence on fusion kinetics to structural features of the protein. At the level of individual E subunits, trimer formation and membrane engagement of the threefold clustered fusion loops are rate-limiting. Hemifusion requires at least two adjacent trimers. Simulation of the kinetics indicates that availability of competent monomers within the contact zone between virus and target membrane makes trimerization a bottleneck in hemifusion. We discuss the implications of the model we have derived for mechanisms of membrane fusion in other contexts.DOI: http://dx.doi.org/10.7554/eLife.04389.001

The orphan receptor tyrosine kinase ErbB2 (HER2/Neu) transforms cells when overexpressed1, and is an important therapeutic target in human cancer2,3. Structural studies4,5 have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular ‘tether’ in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor6. Although ErbB2 is clearly unique among the four human ErbB receptors6,7, we show here that it is the closest structural relative of the single EGF receptor family member (dEGFR) in Drosophila melanogaster. Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands8, yet a crystal structure shows that it too lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches to target novel aspects of this orphan receptor.

SUMMARY Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation in the cytokine erythropoietin (EPO, R150Q). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor, but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.