Papers by Michael Overduin
Biomolecular NMR assignments, 2011
The pleckstrin homology domain of the FAPP1 protein (FAPP1-PH) recognizes phosphatidylinositol 4-... more The pleckstrin homology domain of the FAPP1 protein (FAPP1-PH) recognizes phosphatidylinositol 4-phosphate [PtdIns(4)P] and is recruited to the Golgi apparatus in order to mediate trafficking to the cell surface. We report the complete (1)H, (13)C and (15)N resonance assignments of the FAPP1-PH in its free state and those induced by PtdIns(4)P or detergent micelles.
Journal of biochemistry and molecular biology, Jan 31, 2005
Dishevelled (Dvl) is a positive regulator of the canonical Wnt signaling pathway, which regulates... more Dishevelled (Dvl) is a positive regulator of the canonical Wnt signaling pathway, which regulates the levels of beta-catenin. The beta-catenin oncoprotein depends upon the association of Dvl and Axin proteins through their DIX domains, and its accumulation directs the expression of specific developmental-related genes at the nucleus. Here, the (1)H, (13)C and (15)N resonances of the human Dishevelled 2 DIX domain are assigned using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. In addition, helical and extended elements are identified based on the NMR data. The results establish a structural context for characterizing the actin and phospholipid interactions and binding sites of this novel domain, and provide insights into its role in protein localization to stress fibers and cytoplasmic vesicles during Wnt signaling.
Science (New York, N.Y.), Jan 28, 1998
Eps15 homology (EH) domains are eukaryotic signaling modules that recognize proteins containing A... more 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.
Biomolecular NMR Assignments, 2009
The human AKAP13 protein contains DH and PH domains, which are responsible for its cell transform... more The human AKAP13 protein contains DH and PH domains, which are responsible for its cell transforming activity. Despite its biomedical importance, the contribution of the PH domain to AKAP13 activity remains unclear and no three dimensional structure is available to date. Here we report the backbone and side chain 1 H, 13 C and 15 N resonance assignments of a 20 kDa construct comprising the uniformly 13 C and 15 N labeled AKAP13-PH domain and an associated helix from the DH domain which is required for its stable expression. Resonance assignment has been achieved using conventional triple resonance experiments; 95% of all back bone resonances and more than 90% of side chain resonances have been successfully assigned. The 1 H, 13 C and 15 N chemical shifts have been deposited in BMRB with accession number of 16195.
Journal of Biological Chemistry, 2014
The Lbc oncoprotein stimulates deregulated GTPase activity in RhoA. Results: Although the Lbc DH ... more The Lbc oncoprotein stimulates deregulated GTPase activity in RhoA. Results: Although the Lbc DH domain can independently activate GTP exchange by RhoA, its PH domain also presents surfaces for DH and activated RhoA interaction. Conclusion: Multiple sites on both structural domains of the Lbc scaffold control RhoA. Significance: New sites for mechanism-based design of modulators of Lbc action are revealed.
The Src homology domains, SH3 and SH2, of Abl protein tyrosine kinase regulate enzymatic activity... more 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'. 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). 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.
Journal of molecular biology, Jan 8, 2015
Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) doma... more Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charge...
The EMBO Journal, 2007
Adaptor proteins play important endocytic roles including recognition of internalization signals ... more Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein-ligand complex. The a þ b structure of the protein reveals an SH3-like topology with a solventexposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis.
Science, 1995
Cadherins are calcium-dependent cell adhesion molecules containing extracellular repeats of appro... more 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.
Science, 2001
Phosphoinositide (PI)-binding domains play critical roles in the intracellular localization of a ... more 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.
Nature Structural & Molecular Biology, 2012
Molecular and Cellular Biology, 2006
Journal of Biomolecular NMR, 1996
E-cadherin is a transmembrane protein that provides CaZ+-dependent cell adhesion to epithelial ce... more E-cadherin is a transmembrane protein that provides CaZ+-dependent cell adhesion to epithelial cells. The large majority of the 1H, 15N, 13C and ~3CO 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 Science, 267, 386-389], has been refined. The monomeric state of this isolated domain is demonstrated by light scattering and sedimentation analysis. Seven [3-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-13-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 13-sheet region and quasi-13-helix. The 13-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 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.
Journal of Biological Chemistry, 2006
The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently ov... more The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.
Journal of Biological Chemistry, 2009
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole ... more The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.
EMBO reports, 2010
This is an open-access article distributed under the terms of the Creative Commons Attribution Li... more This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission.
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Papers by Michael Overduin