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M Overduin

Publications and source records attributed to M Overduin.

At least 19 recordsLinked to original sources
PubMed2001

Location, location, location: membrane targeting directed by PX domains.

Phosphoinositide (PI)-binding domains play critical roles in the intracellular localization of a variety of cell-signaling proteins. The 120-amino acid Phox homology (PX) domain targets proteins to organelle membranes through interactions between two conserved basic motifs within the PX domain and specific PIs. The combination of protein-lipid and protein-protein interactions ensures the proper localization and regulation of PX domain-containing proteins. Upon proper localization, PX domain-containing proteins can then bind to additional proteins and execute their functions in a diverse set of biological pathways, including intracellular protein transport, cell growth and survival, cytoskeletal organization, and neutrophil defense.

Amino Acid Motifs↗
PubMed2001

Phosphatidylethanolamine has an essential role in Saccharomyces 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 approximately 90% of total cellular phosphatidylserine decarboxylase activity. When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta 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 psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine. In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Delta psd2Delta 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 by (31)P NMR.

Chromatography, Liquid↗
PubMed2001

Structural mechanism of endosome docking by the FYVE domain.

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.

Binding Sites↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Signaling with phosphoinositides: better than binary.

When the outside of cell is stimulated,the inside generates a flurry of signals. Phosphates are sprinkled over lipids and proteins,where they are recognized within diverse signaling pathways. The kinases that congregate beneath the cell surface to provide the phosphate tags that mediate signaling have become major targets of new wave of drug design. Phosphoinositide signaling presents a particularly intriguing network whose many mysteries are now being unlocked. Research into protein domains that specifically recognize phosphoinositides have established the ENTH, FYVE,Phox,and pleckstrin homology domains s four cornerstones of phosphoinositide signaling.

1-Phosphatidylinositol 4-Kinase↗
PubMed2000

Solution structure of Eps15's third EH domain reveals coincident Phe-Trp and Asn-Pro-Phe binding sites.

Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Molecular mechanism of NPF recognition by EH domains.

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.

Amino Acid Motifs↗
PubMed1999

Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

Terminase is an enzyme from bacteriophage lambda that is required for insertion of the viral genome into an empty pro-capsid. This enzyme is composed of the viral proteins gpNu1 (20.4 kDa) and gpA (73.3 kDa) in a holoenzyme complex. Current models for terminase assembly onto DNA suggest that gpNu1 binds to three repeating elements within a region of the lambda genome known as cosB which, in turn, stimulates the assembly of a gpA dimer at the cosN subsite. This prenicking complex is the first of several stable nucleoprotein intermediates required for DNA packaging. We have noted a hydrophobic region within the primary amino acid sequence of the terminase gpNu1 subunit and hypothesized that this region constitutes a protein-protein interaction domain required for cooperative assembly at cosB and that is also responsible for the observed aggregation behavior of the isolated protein. We therefore constructed a mutant of gpNu1 in which this hydrophobic "domain" has been deleted in order to test these hypotheses. The deletion mutant protein, gpNu1DeltaK, is fully soluble and, unlike full-length protein, shows no tendency toward aggregation; However, the protein is a dimer under all experimental conditions examined as determined by gel permeation and sedimentation equilibrium analysis. The truncated protein is folded with evidence of secondary and tertiary structural elements by circular dichroism and NMR spectroscopy. While physical and biological assays demonstrate that gpNu1DeltaK does not interact with the terminase gpA subunit, the deletion mutant binds with specificity to cos-containing DNA. We have thus constructed a deletion mutant of the phage lambda terminase gpNu1 subunit which constitutes a highly soluble DNA binding domain of the protein. We further propose that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

Bacteriophage lambda↗
PubMed1999

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 domain's 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.

Amino Acid Sequence↗
PubMed1998

Structure and Asn-Pro-Phe binding pocket of the Eps15 homology domain.

Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing Asn-Pro-Phe (NPF) sequences. The structure of the central EH domain of Eps15 has been solved by heteronuclear magnetic resonance spectroscopy. The fold consists of a pair of EF hand motifs, the second of which binds tightly to calcium. The NPF peptide is bound in a hydrophobic pocket between two alpha helices, and binding is mediated by a critical aromatic interaction as revealed by structure-based mutagenesis. The fold is predicted to be highly conserved among 30 identified EH domains and provides a structural basis for defining EH-mediated events in protein trafficking and growth factor signaling.

Amino Acid Sequence↗
PubMed1996

Structural basis of calcium-induced E-cadherin rigidification and dimerization.

The cadherins mediate cell adhesion and play a fundamental role in normal development. They participate in the maintenance of proper cell-cell contacts: for example, reduced levels of epithelial cadherin (E-cadherin) correlate with increased invasiveness in many human tumour cell types. The cadherins typically consist of five tandemly repeated extracellular domains, a single membrane-spanning segment and a cytoplasmic region. The N-terminal extracellular domains mediate cell-cell contact while the cytoplasmic region interacts with the cytoskeleton through the catenins. Cadherins depend on calcium for their function: removal of calcium abolishes adhesive activity, renders cadherins vulnerable to proteases (reviewed in ref. 4) and, in E-cadherin, induces a dramatic reversible conformational change in the entire extracellular region. We report here the X-ray crystal structure at 2.0 A resolution of the two N-terminal extracellular domains of E-cadherin in the presence of calcium. The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions. Not only do the bound calcium ions linearize and rigidify the molecule, they promote dimerization. Although the N-terminal domain of each molecule in the dimer is aligned in a parallel orientation, the interactions between them differ significantly from those found in the neural cadherin (N-cadherin) N-terminal domain (NCD1) structure. The E-cadherin dual-domain structure reported here defines the role played by calcium in the cadherin-mediated formation and maintenance of solid tissues.

Amino Acid Sequence↗
PubMed1996

1H, 15N and 13C resonance assignments and monomeric structure of the amino-terminal extracellular domain of epithelial cadherin.

E-cadherin is a transmembrane protein that provides Ca(2+)-dependent cell adhesion to epithelial cells. The large majority of the 1H, 15N, 13C and 13CO resonances of a 146-amino acid polypeptide from epithelial (E-) cadherin have been assigned using multidimensional NMR spectroscopy. The structure of the amino-terminal 100 amino acids, corresponding to the first extracellular repeat of E-cadherin [Overduin et al. (1995) Science, 267, 386-389], has been refined. The monomeric state of this isolated domain is demonstrated by light scattering and sedimentation analysis. Seven beta-strands and two short helices were identified by patterns of NOE cross-peaks, vicinal coupling constants and chemical shift indices. A novel structural motif termed a quasi-beta-helix found in the crystal structure of a neural (N-) cadherin domain [Shapiro et al. (1995) Nature, 374, 327-337] is characterized in detail for the first time by NMR. Slowly exchanging amides were concentrated in the beta-sheet region and quasi-beta-helix. The beta-barrel fold of the cadherin domain is topologically similar to the immunoglobulin fold. Comparison of this solution structure to the crystallized dimers of the N-terminal pair of E-cadherin domains [Nagar et al. (1996) Nature, 380, 360-364] and of the homologous single domain of N-cadherin reveals a conserved cadherin fold with minor structural differences, which can be accounted for by differences in metal ligation and oligomeric state.

Amino Acid Sequence↗
PubMed1995

The solution structure of Abl SH3, and its relationship to SH2 in the SH(32) construct.

BACKGROUND: The Src homology domains, SH3 and SH2, of Abl protein tyrosine kinase regulate enzymatic activity in vivo. Abl SH3 suppresses kinase activity, whereas Abl SH2 is required for the transforming activity of the activated form of Abl. We expect that the solution structures of Abl SH3, Abl SH2 and Abl SH(32) (a dual domain comprising SH3 and SH2 subdomains) will contribute to a structural basis for understanding the mechanism of the Abl 'regulatory apparatus'. RESULTS: We present the solution structure of the free Abl SH3 domain and a structural characterization of the Abl regulatory apparatus, the SH(32) dual domain. The solution structure of Abl SH3 was determined using multidimensional double resonance NMR spectroscopy. It consists of two antiparallel beta sheets packed orthogonally, an arrangement first shown in spectrin SH3. Compared with the crystal structure of the Abl SH3 complexed with a natural ligand, there is no significant difference in overall folding pattern. The structure of the Abl SH(32) dual domain was characterized by NMR spectroscopy using the 1H and 15N resonance assignment of Abl SH3 and Abl SH2. On the basis of the high degree of similarity in chemical shifts and hydrogen/deuterium exchange pattern for the individual domains of SH3 and SH2 compared with those of the SH(32) dual domain, a structural model of the Abl SH(32) regulatory apparatus is suggested. This model is in good agreement with the ligand-binding characteristics of Abl SH3, SH2 and SH(32). The binding constants for isolated SH3 and SH2 domains when binding to natural ligands, measured by intrinsic fluorescence quenching, do not differ significantly from the constants of these domains within SH(32). CONCLUSION: The solution structures of free Abl SH3 and Abl SH2, and the structural model of Abl SH(32), provide information about the overall topology of these modular domains. The structural model of Abl SH(32), a monomer, consists of the SH3 and SH2 domains connected by a flexible linker. Sites of ligand binding for the two subdomains are independent.

Amino Acid Sequence↗
PubMed1995

Solution structure of the epithelial cadherin domain responsible for selective cell adhesion.

Cadherins are calcium-dependent cell adhesion molecules containing extracellular repeats of approximately 110 amino acids. The three-dimensional structure of the amino-terminal repeat of mouse epithelial cadherin was determined by multidimensional heteronuclear magnetic resonance spectroscopy. The calcium ion was bound by a short alpha helix and by loops at one end of the seven-stranded beta-barrel structure. An exposed concave face is in a position to provide homophilic binding specificity and was also sensitive to calcium ligation. Unexpected structural similarities with the immunoglobulin fold suggest an evolutionary relation between calcium-dependent and calcium-independent cell adhesion molecules.

Amino Acid Sequence↗