Michael Overduin

Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes

Specific recognition of phosphoinositides is crucial for protein sorting and membrane trafficking. Protein transport to the yeast vacuole depends on the Vam7 t-SNARE and its phox homology (PX) domain. Here, we show that the PX domain of Vam7 targets to vacuoles in vivo in a manner dependent on phosphatidylinositol 3-phosphate generation. A novel phosphatidylinositol-3-phosphate-binding motif and an exposed loop that interacts with the lipid bilayer are identified by nuclear magnetic resonance spectroscopy. Conservation of key structural and binding site residues across the diverse PX family indicates a shared fold and phosphoinositide recognition function.

The DIX domain targets dishevelled to actin stress fibres and vesicular membranes

Colorectal cancer results from mutations in components of the Wnt pathway that regulate β-catenin levels. Dishevelled (Dvl or Dsh) signals downstream of Wnt receptors and stabilizes β-catenin during cell proliferation and embryonic axis formation. Moreover, Dvl contributes to cytoskeletal reorganization during gastrulation and mitotic spindle orientation during asymmetric cell division. Dvl belongs to a family of eukaryotic signalling proteins that contain a conserved 85-residue module of unknown structure and biological function called the DIX domain. Here we show that the DIX domain mediates targeting to actin stress fibres and cytoplasmic vesicles in vivo. Neighbouring interaction sites for actin and phospholipid are identified between two helices by nuclear magnetic resonance spectroscopy (NMR). Mutation of the actin-binding motif abolishes the cytoskeletal localization of Dvl, but enhances Wnt/β-catenin signalling and axis induction in Xenopus. By contrast, mutation of the phospholipid interaction site disrupts vesicular association of Dvl, Dvl phosphorylation, and Wnt/β-catenin pathway activation. We propose that partitioning of Dvl into cytoskeletal and vesicular pools by the DIX domain represents a point of divergence in Wnt signalling.

Phosphatidylinositol 3-Phosphate Recognition by the FYVE Domain

Recognition of phosphatidylinositol 3-phosphate (Ptdlns(3)P) is crucial for a broad range of cellular signaling and membrane trafficking events regulated by phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion, involves two beta hairpins and an alpha helix. Specific amino acids, including those of the FYVE domains conserved RRHHCRQCGNIF motif, contact soluble and micelle-embedded lipid and provide specificity for Ptdlns(3)P over Ptdlns(5)P and Ptdlns, as shown by heteronuclear magnetic resonance spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by ovel structural features and its ptdlns(3)P-binding site.

Phosphatidylethanolamine Has an Essential Role inSaccharomyces cerevisiae That Is Independent of Its Ability to Form Hexagonal Phase Structures

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides ∼90% of total cellular phosphatidylserine decarboxylase activity. When thePSD1 gene is deleted, the resultant strain(psd1Δ) grows normally at 30u2009°C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37u2009°C) or on the nonfermentable carbon source lactate, the growth of psd1Δ strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Δ psd2Δ dpl1Δ strain can only grow in the presence of ethanolamine. In contrast, a psd1Δ psd2Δstrain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Δ psd2Δ cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by31P NMR.

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.

Insights into specific DNA recognition during the assembly of a viral genome packaging machine.

Terminase enzymes mediate genome packaging during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy. Its fold contains a unique winged helix-turn-helix (wHTH) motif within a novel scaffold. Surprisingly, a predicted P loop ATP binding motif is in fact the wing of the DNA binding motif. Structural and genetic analysis has identified determinants of DNA recognition specificity within the wHTH motif and the DNA recognition sequence. The structure reveals an unexpected DNA binding mode and provides a mechanistic basis for the concerted action of gpNu1 and Escherichia coli integration host factor during assembly of the packaging machinery.

The plot thickens: how thrombin modulates blood clotting.

The tide of the blood coagulation cascade turns by a switch in thrombin specificity. Two new structures reveal how thrombomodulins EGF domains flip the switch by blocking old substrates and docking new ones.

In support of the BMRB.

volume 19 number 9 SePTember 2012 nature structural & molecular biology of intrinsically disordered proteins, development of automated analysis of NMR data, solid-state NMR and NMR-based metabolomics. With the budget cuts that the BMRB has suffered (reduced by 40%, compared to the previous operating budget), we currently are at the minimal level of keeping up with depositions, data validation and datadictionary development. In addition, the BMRB is barely managing to meet its obligations as a partner in the wwPDB. We have had to lay off people who were developing new software and functionality. The wwPDB advisory-committee meeting, held at Rutgers University on 1 October 2010, had a session on funding, which enabled us to inform members of the US granting agencies about the impending expiration of remaining funding from the National Library of Medicine in September 2014. To date, no plan has been advanced to keep the BMRB functioning. None of the three agencies has expressed an interest in funding more than a part of the needed budget, so a multiagency approach appears to be needed. To stimulate this, BMRB staff members prepared a ‘white paper’ (see Supplementary Note), which was approved by its advisory board and then sent to representatives of the US grant agencies (National Institutes of Health (NIH), Department of Energy and National Science Foundation). Given the lead time for applications and review, it appears critical that a funding plan be developed within the coming year.