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1.  Lipid binding promotes oligomerization and focal adhesion activity of vinculin 
The Journal of Cell Biology  2014;207(5):643-656.
PIP2 binds vinculin and directs its oligomerization, which promotes proper focal adhesion structure and function.
Adherens junctions (AJs) and focal adhesion (FA) complexes are necessary for cell migration and morphogenesis, and for the development, growth, and survival of all metazoans. Vinculin is an essential regulator of both AJs and FAs, where it provides links to the actin cytoskeleton. Phosphatidylinositol 4,5-bisphosphate (PIP2) affects the functions of many targets, including vinculin. Here we report the crystal structure of vinculin in complex with PIP2, which revealed that PIP2 binding alters vinculin structure to direct higher-order oligomerization and suggests that PIP2 and F-actin binding to vinculin are mutually permissive. Forced expression of PIP2-binding–deficient mutants of vinculin in vinculin-null mouse embryonic fibroblasts revealed that PIP2 binding is necessary for maintaining optimal FAs, for organization of actin stress fibers, and for cell migration and spreading. Finally, photobleaching experiments indicated that PIP2 binding is required for the control of vinculin dynamics and turnover in FAs. Thus, through oligomerization, PIP2 directs a transient vinculin sequestration at FAs that is necessary for proper FA function.
PMCID: PMC4259812  PMID: 25488920
2.  The metavinculin tail domain directs constitutive interactions with raver1 and vinculin RNA 
Journal of molecular biology  2012;422(5):10.1016/j.jmb.2012.06.015.
Vinculin is a key regulator of the attachment of the actin cytoskeleton to the cell membrane at cellular adhesion sites that is crucial for processes like cell motility and migration, development, survival, and wound healing. Vinculin loss results in embryonic lethality, cardiovascular diseases, and cancer. Its tail domain, Vt, is crucial for vinculin activation and focal adhesion turnover and binds to the actin cytoskeleton and acidic phospholipids upon which it unfurls. The RNA binding protein raver1 regulates the assembly of focal adhesions transcriptionally by binding to vinculin. The muscle-specific splice form, metavinculin, is characterized by a 68 residue insert in the tail domain (MVt) and correlates with hereditary idiopathic dilated cardiomyopathy. Here we report that metavinculin can bind to raver1 in its inactive state. Our crystal structure explains this permissivity, where an extended coil unique to MVt is unfurled in the MVtΔ954:raver1 complex structure. Our binding assays show that raver1 forms a ternary complex with MVt and vinculin mRNA. These findings suggest that the metavinculin:raver1:RNA complex is constitutively recruited to adhesion complexes.
PMCID: PMC3835166  PMID: 22709580
adherens junction; cardiomyopathy; focal adhesion; RRM domain; RNA binding
3.  Dimer asymmetry defines α-catenin interactions 
The F-actin binding cytoskeletal protein α-catenin interacts with β-catenin-cadherin complexes and stabilizes cell-cell junctions. The β-catenin–α-catenin complex cannot bind to F-actin, whereas interactions of α-catenin with the cytoskeletal protein vinculin appear necessary to stabilize adherens junctions. Here we report the crystal structure of nearly full-length human α-catenin at 3.7 Å resolution. α-Catenin forms an asymmetric dimer, where the four-helix bundle domains of each subunit engage in distinct intermolecular interactions. This results in a left handshake-like dimer, where the two subunits have remarkably different conformations. The crystal structure explains why dimeric α-catenin has a higher affinity for F-actin than monomeric α-catenin, why the β-catenin–α-catenin complex does not bind to F-actin, how activated vinculin links the cadherin-catenin complex to the cytoskeleton, and why α-catenin but not inactive vinculin can bind to F-actin.
PMCID: PMC3805043  PMID: 23292143
4.  Improving the diffraction of full-length human selenomethionyl metavinculin crystals by streak-seeding 
Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of dilated-cardiomyopathy-associated mutant metavinculin crystals.
Metavinculin is an alternatively spliced isoform of vinculin that has a 68-residue insert in its tail domain (1134 total residues) and is exclusively expressed in cardiac and smooth muscle tissue, where it plays important roles in myocyte adhesion complexes. Mutations in the metavinculin-specific insert are associated with dilated cardiomyopathy (DCM) in man. Crystals of a DCM-associated mutant of full-length selenomethionine-labeled metavinculin grown by hanging-drop vapor diffusion diffracted poorly and were highly sensitive to radiation, preventing the collection of a complete X-ray diffraction data set at the highest possible resolution. Streak-seeding markedly improved the stability, crystal-growth rate and diffraction quality of DCM-associated mutant metavinculin crystals, allowing complete data collection to 3.9 Å resolution. These crystals belonged to space group P43212, with two molecules in the asymmetric unit and unit-cell parameters a = b = 170, c = 211 Å, α = β = γ = 90°.
PMCID: PMC2998368  PMID: 21139209
metavinculin; streak-seeding
5.  Dramatic improvement of crystal quality for low-­temperature-grown rabbit muscle aldolase 
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination.
Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA–LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 Å Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA–LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number of interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice.
PMCID: PMC2864701  PMID: 20445268
rabbit muscle aldolase; improvement of crystal quality; low-complexity domain; sorting nexin 9
6.  Raver1 interactions with Vinculin and RNA Suggest a Feed-Forward Pathway in Directing mRNA to Focal Adhesions 
The translational machinery of the cell re-localizes to focal adhesions following the activation of integrin receptors. This response allows for rapid, local production of components needed for adhesion complex assembly and signaling. Vinculin links focal adhesions to the actin cytoskeleton following its activation by integrin signaling, which severs intramolecular interactions of the vinculin head and tail (Vt) domains. Our vinculin:raver1 crystal structures and binding studies show that activated Vt selectively interacts with one of the three RNA recognition motifs (RRM) of raver1, that the vinculin:raver1 complex binds to F-actin, and that raver1 binds selectively to RNA, including a sequence found in vinculin mRNA. Further, mutation of residues that mediate interaction of raver1 with vinculin abolish their co-localization in cells. These findings suggest a feed-forward model where vinculin activation at focal adhesions provides a scaffold for recruitment of raver1 and its mRNA cargo to facilitate the production of components of adhesion complexes.
PMCID: PMC2811071  PMID: 19523901
focal adhesion; actin cytoskeleton; crystallography; RNP motif; RNA binding
7.  A Helix Replacement Mechanism Directs Metavinculin Functions 
PLoS ONE  2010;5(5):e10679.
Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated ΔLeu954 metavinculin deletion mutant. These structures reveal that an α-helix (H1′) and extended coil of the metavinculin insert replace α-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated ΔLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1′ α-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.
PMCID: PMC2873289  PMID: 20502710
8.  Structure of [NiFe] Hydrogenase Maturation Protein HypE from Escherichia coli and Its Interaction with HypF▿  
Journal of Bacteriology  2007;190(4):1447-1458.
Hydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-Å resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of ∼400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.
PMCID: PMC2238214  PMID: 18065529
9.  Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis 
Journal of Bacteriology  2006;188(15):5606-5617.
Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence →3)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.
PMCID: PMC1540030  PMID: 16855251

Results 1-9 (9)