Structure - Activities
Some Migration-Related Structures & New Methodologies
| Alpha-Actinin | Actin-Fimbrin Complexes | Arp2/3 Complex and Actin Networks |
| Actin-Myosin Complexes | Collagen-Integrin Complex | Talin-Integrin Complex |
Cryo-Em structure of chicken gizzard smooth muscle alpha-actinin
Cryoelectron microscopy was used to obtain a 3-D image at 2.0 nm resolution of 2-D arrays of smooth muscle alpha-actinin. The reconstruction reveals a well-resolved long central domain with 90 degrees of left-handed twist and near 2-fold symmetry. However, the molecular ends which contain the actin binding and calmodulin-like domains, have different structures oriented approximately 90 degrees to each other. Atomic structures for the alpha-actinin domains were built by homology modeling and assembled into an atomic model. Model building suggests that in the 2-D arrays, the two calponin homology domains that comprise the actin-binding domain have a closed conformation at one end and an open conformation at the other end due to domain swapping. The open and closed conformations of the actin-binding domain suggests flexibility that may underlie Ca2+ regulation. The approximately 90 degrees orientation difference at the molecular ends may underlie alpha-actinin's ability to crosslink actin filaments in nearly any orientation.
Liu J, Taylor DW, Taylor KA. A 3-D reconstruction of smooth muscle
alpha-actinin by CryoEm reveals two different conformations at the
actin-binding region. J Mol Biol. 2004 Apr 16;338(1):115-25.
PubMed;
Other related PDB entries;
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Crystal Structure Of The Rod Domain Of Alpha-Actinin.
Ylanne J, Scheffzek K, Young P, Saraste M. Structure (Camb).
2001 Jul 3;9(7):597-604.
PubMed;
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Solution Structure Of Pdz Domain Of Mouse Alpha-Actinin 2
Associated Lim Protein. In press.
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Calponin Homology (Ch) Domain From Human Beta-Spectrin.
Carugo KD, Banuelos S, Saraste M. Nat Struct Biol. 1997
Mar;4(3):175-9.
PubMed;
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Ef-Hands 3,4 From Alpha-Actinin Z-Repeat 7 From Titin.
Atkinson RA, Joseph C, Kelly G, Muskett FW, Frenkiel TA,
Nietlispach D, Pastore A. Nat Struct Biol. 2001
Oct;8(10):853-7.
PubMed;
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Crystal Structure Of Two Central Spectrin-Like Repeats From
Alpha-Actinin. Djinovic-Carugo K, Young P, Gautel M,
Saraste M. Cell. 1999 Aug 20;98(4):537-46.
PubMed;
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An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation. Using a new procedure that combines electron-density correlation with biochemical information, we have fitted the crystal structure of the N-terminal actin-binding domain of human T-fimbrin to helical reconstructions of fimbrin-decorated actin filaments. The map locates the N-terminal calcium-binding domain and identifies actin-binding site residues on the two calponin-homology domains of fimbrin. Based on this map, we propose a model of a fimbrin crosslink in an actin bundle and its regulation by calcium. Hanein D, Volkmann N, Goldsmith S, Michon AM, Lehman W, Craig R, DeRosier D, Almo S, Matsudaira P. An atomic model of fimbrin binding to F-actin and its implications for filament crosslinking and regulation. Nat Struct Biol. 1998 Sep;5(9):787-92. PubMed An atomic model of actin filaments cross-linked by fimbrin and its implications for bundle assembly and function. Actin bundles have profound effects on cellular shape, division, adhesion, motility, and signaling. Fimbrin belongs to a large family of actin-bundling proteins and is involved in the formation of tightly ordered cross-linked bundles in the brush border microvilli and in the stereocilia of inner ear hair cells. Polymorphism in these three-dimensional (3D) bundles has prevented the detailed structural characterization required for in-depth understanding of their morphogenesis and function. Here, we describe the structural characterization of two-dimensional arrays of actin cross-linked with human T-fimbrin. Structural information obtained by electron microscopy, x-ray crystallography, and homology modeling allowed us to build the first molecular model for the complete actin-fimbrin cross-link. The restriction of the arrays to two dimensions allowed us to deduce the spatial relationship between the components, the mode of fimbrin cross-linking, and the flexibility within the cross-link. The atomic model of the fimbrin cross-link, the cross-linking rules deduced from the arrays, and the hexagonal packing of actin bundles in situ were all combined to generate an atomic model for 3D actin-fimbrin bundles. Furthermore, the assembly of the actin-fimbrin arrays suggests coupling between actin polymerization, fimbrin binding, and crossbridge formation, presumably achieved by a feedback between conformational changes and changes in affinity. |
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Figure: Atomic models of 2D actin arrays cross-linked by fimbrin. The spatial relationships between the filaments for the two types of arrays were deduced from the observed diffraction patterns. The filaments in the micrographs with straight crossbands are in register. The filaments in the micrographs with slanted crossbands are rotated by 26.66° in respect to their neighbors. This operation exposes the same actin interface only one notch down . (A-C) Model of the straight crossband with filaments in register. Actin is shown in gray, ABD1 in pink, ABD2 in blue, and the N-terminal calcium-binding domain (EF-hands) in cyan. The spatial relationship between the filaments and the ABDs was taken from the atomic model of actin-ABD1. The position of the calcium-binding domain was deduced from the difference peak between the docked model and the observed 3D reconstruction. ABD2 and ABD1 could be exchanged in principle; however, steric clashes between ABD2 and the calcium-binding domain would result if we exchange the ABD1 with ABD2. (A) Shows a side view of the arrays (as seen by the microscope). The actin monomers are enumerated, crossbridges occur at positions 0, 13, and 26. (B) Shows a view looking down the filament axes. The lipid layer is located on top of the figure. (C) A magnified view of a cross-link. (D) Fourier transform of the 2D array in A. (E) Overlay of an enhanced version of an observed micrograph and a scaled version (light gray) of the atomic model in A. Note how well the crossband and filament distances correspond. (F-G and J) Model of the slanted crossband with adjacent filaments rotated by 26.66°, equivalent to a 55-Å downward translation. This geometry can be achieved by rotating fimbrin by 180° around its center of mass parallel to the filament axes and accommodating for the slight offset of fimbrin's geometry from exact rotational geometry. This symmetry is immediately evident by comparing B and F. Note the seemingly different position of the calcium-binding domain (C and G) is due to slightly different views. (F) A view looking down the filaments. The lipid layer is located on top of the figure. The criterion for handedness in this arrangement would be a preference for maximum distance from the lipid layer. (J) A side view, and (G) a magnified view of a cross-link. (H) Overlay of an enhanced version of an observed micrograph and a scaled version (light gray) of the atomic model in J. Note how well the crossband and filament distances correspond. (I) Fourier transform of the 2D array in J. Volkmann N, DeRosier D, Matsudaira P, Hanein D. An atomic model of actin filaments cross-linked by fimbrin and its implications for bundle assembly and function. J Cell Biol. 2001 May 28;153(5):947-56. PubMed Other related PDB entries;
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Arp2/3 Complex and Actin Networks
Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of Acanthamoeba castellanii and Saccharomyces cerevisiae Arp2/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch initiation involves large-scale structural rearrangements within Arp2/3. |
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Figure:
Molecular model of the actin-bound Arp2/3 complex at the branch junction.
(A) Model of actin filament branches mediated by Acanthamoeba Arp2/3
complex. The backbone of the molecular model of filamentous actin,
fitted to the 2D reconstruction, is shown in pink. The first two subunits
of the daughter filament, shown in red and green backbone presentation,
are assigned to be Arps. The other five subunits of the complex are
assigned to the rest of the projection density using the proximity
information from cross-linking, genetic, and yeast two-hybrid experiments:
p40 (purple); p35 (pink); p18 (yellow); p19 (light blue); p14 (orange).
The size of the regions was chosen to approximate the respective molecular
weights, assuming a thickness of about 5 nm. The barbed ends of the
filaments are toward the top of the figure. (B) Images of Arp2/3 complex
from Acanthamoeba bound to the side of mother filaments. All samples
in the actin experiments were prepared in the presence of the activator
(WA). Upper row: in vitrified buffer, a Gaussian real-space filter
was applied to the images for visualization. Lower row: quick-frozen,
deep-etched, rotary-shadowed specimen. Note the similarity of the
position and shape of the complex to that seen at the branch junction
shown in (A). Bar is 7 nm. (C) Average of six aligned images of Arp2/3
complex bound to the side of filaments in vitrified buffer overlaid
with backbone presentation of an actin filament (pink) and backbone
presentations of Arp2 (red) and Arp3 (green) in positions similar
to those in (A). The remaining density was assigned to the other subunits
and colored as in (A). (D) Density representations of the models of
actin-bound (green) and the free, WA-activated Arp2/3 complex. The
density for the branched model was calculated using a filament-like
configuration for the two Arps and using the remaining projection
density assuming a thickness of ~5 nm. The view was generated by turning
the model of the complex in (A) by 90° counter-clockwise. On the
right, the best fit of the density representing the branched model
(green) into the 3D reconstruction of the free Arp2/3 complex (gray)
is shown. Note that the density corresponding to Arp2 cannot be accommodated
by the reconstruction of the free complex. A possible large-scale
conformational change of the free, activated complex upon binding
to actin, a rearrangement in the position of Arp2, is indicated by
an arrow. (E) The density for the branched model (green, left) and
the best fit (right) of the branched model density (green) into that
of the free Arp2/3 complex (gray). The binding site of the WA N-terminus
(gold), as assigned by the labeling. With this fit the WA N-terminus
is in close proximity to the F-actin interface of the Arp2/3 complex.
The orientation of this view matches that of the projection densities
in (A) and (C). A possible rearrangement [as in (D)] of Arp2 upon
binding to the filament is indicated by an arrow. (F) The best fit
of the density of the branched model (green) into that of the free
Arp2/3 complex (gray). WA N-terminus is shown in gold. The straight
and circular arrows on the right indicate the axis and turning direction
that generate this view from the view in (E).
Volkmann N, Amann KJ, Stoilova-McPhie S, Egile C, Winter DC, Hazelwood L, Heuser JE, Li R, Pollard TD, Hanein D. Structure of Arp2/3 complex in its activated state and in actin filament branch junctions. Science. 2001 Sep 28;293(5539):2456-9. PubMed Other related PDB entries;
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Actin-Myosin Complexes
Evidence for cleft closure in actomyosin upon ADP release. Structural insights into the interaction of smooth muscle myosin with actin have been provided by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy, and by mapping of structural and dynamic changes in the actomyosin complex. The actomyosin structures determined in the presence and absence of MgADP differ significantly from each other, and from all crystallographic structures of unbound myosin. Coupled to a complex movement ( approximately 34 A) of the light chain binding domain upon MgADP release, we observed a approximately 9 degrees rotation of the myosin motor domain relative to the actin filament, and a closure of the cleft that divides the actin binding region of the myosin head. Cleft closure is achieved by a movement of the upper 50 kDa region, while parts of the lower 50 kDa region are stabilized through strong interactions with actin. This model supports a mechanism in which binding of MgATP at the active site opens the cleft and disrupts the interface, thereby releasing myosin from actin. |
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Figure:
Best fits for crystal structures to the 3D rigor reconstructions.
a, smooth muscle S1 (transition state), b, skeletal S1 (nucleotide
free) and c, scallop S1 (in the presence of ADP) into a reconstruction
of S1 decorated actin in the rigor state. The RLC for the smooth muscle
S1 was modeled after the skeletal S1 structure. d, The fit for the
final model. The ELC is shown in red, the RLC in light blue and the
converter in orange. Only an envelope that corresponds to a single
S1 molecule is shown. The contour level was chosen to enclose only
significant density at a 99.5% confidence level. The fits for the
crystal structures are dominated by the motor domain while most of
the light chain domains are placed outside the significant density.
The actin binding sites are located at the right side of the figures,
the light chain domain to the left. The arrow (a) points toward the
sarcomeric M line (up in Fig. 1) and coincides with the F-actin axis.
Volkmann N, Hanein D, Ouyang G, Trybus KM, DeRosier DJ, Lowey S. Evidence for cleft closure in actomyosin upon ADP release. Nat Struct Biol. 2000 Dec;7(12):1147-55. PubMed
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Myosin isoforms show unique conformations in the actin-bound state.
Crystallographic data for several myosin isoforms have provided evidence for at least two conformations in the absence of actin: a prehydrolysis state that is similar to the original nucleotide-free chicken skeletal subfragment-1 (S1) structure, and a transition-state structure that favors hydrolysis. These weak-binding states differ in the extent of closure of the cleft that divides the actin-binding region of the myosin and the position of the light chain binding domain or lever arm that is believed to be associated with force generation. Previously, we provided insights into the interaction of smooth-muscle S1 with actin by computer-based fitting of crystal structures into three-dimensional reconstructions obtained by electron cryomicroscopy. Here, we analyze the conformations of actin-bound chicken skeletal muscle S1. We conclude that both myosin isoforms in the nucleotide-free, actin-bound state can achieve a more tightly closed cleft, a more downward position of the lever arm, and more stable surface loops than those seen in the available crystal structures, indicating the existence of unique actin-bound conformations. |
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Figure:
(a and b) Three-dimensional reconstructions of actin filaments decorated
with skeletal S1. The final skeletal S1 models and the corresponding
3D reconstructions of actomyosin in the presence of MgADP (a) and
in the absence of nucleotide (b) are shown. The contour levels were
chosen to enclose only significant density at a 99.5% confidence level.
Two neighboring molecules are shown: motor domain in green and ELC
in red. All presentations are with the sarcomeric M line at the top
of the figure. Note that there is no detectable rearrangement. (c
and d) Comparison of ELC positions of actin-bound smooth and skeletal
S1. Molecular surface representations of the fitted models, calculated
at 15-Å resolution, are shown. The view in d is rotated by 90°
in respect to the view in c. There is a pronounced difference in the
position of the smooth-muscle ELC in the presence of ADP (magenta)
and the absence of nucleotide (green). There is no such difference
for the skeletal ELC (light blue). Only the motor domain of skeletal
myosin is shown (light gray). The smooth-muscle motor domain rotates
by 9° around the axis shown in red (solid gray lines in d). There
is no significant rearrangement of the skeletal motor domain that
is positioned about halfway between the two smooth-muscle actin-bound
structures (dashed gray line in d).
Volkmann N, Ouyang G, Trybus KM, DeRosier DJ, Lowey S, Hanein D. Myosin isoforms show unique conformations in the actin-bound state. Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3227-32. PubMed Other related PDB entries;
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Collagen-Integrin Complex
Structural basis of collagen recognition by integrin alpha2beta1.
We have determined the crystal structure of a complex between the
I domain of integrin alpha2beta1 and a triple helical collagen peptide
containing a critical GFOGER motif. Three loops on the upper surface
of the I domain that coordinate a metal ion also engage the collagen,
with a collagen glutamate completing the coordination sphere of
the metal. Comparison with the unliganded I domain reveals a change
in metal coordination linked to a reorganization of the upper surface
that together create a complementary surface for binding collagen.
Conformational changes propagate from the upper surface to the opposite
pole of the domain, suggesting both a basis for affinity regulation
and a pathway for signal transduction. The structural features observed
here may represent a general mechanism for integrin-ligand recognition. |
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Figure:
Structure of the I Domain:Collagen Complex (A) Stereo diagram of the
alpha2-I domain in complex with the collagen peptide. The I domain
helices are shown as cylinders, beta-strands as arrows. The three
strands of the collagen triple helix are shown as colored ribbons:
leading strand in green, middle strand in yellow, and trailing strand
in blue.(B) Close-up of A, showing details of the I domain collagen
interface. Selected side chains are shown as ball-and-stick, with
H bonds as dotted lines. The metal ion is shown as a blue ball labeled
"M." The principal interactions with the middle strand GFOGER
motif (yellow) are: phenylalanine makes van der Waals contacts with
side chains of N154 and Q215; the hydroxyproline carbonyl hydrogen
bonds to N154; the glutamate bonds to the metal and H-bonds to T221;
the arginine side chain salt bridges to D215, while its carbonyl H-bonds
to H258. The principal interactions with the trailing strand GFOGER
motif (blue) are: the main chain carbonyl preceding the GFOGER motif
H-bonds to Y157; the phenylalanine makes van der Waals contacts with
L286 and Y157; the hydroxyproline H-bonds to N154 main chain; the
arginine makes weak ionic interactions with E256. The leading strand
(green) makes no contacts with the I domain.(C) Stereo diagram of
the MIDAS motif. The metal ion is shown as a blue ball. Coordinating
side chains are shown as ball-and-stick, with oxygen atoms in red,
carbon in black. Water molecules are labeled "omega"; the
collagen glutamate is in gold. The three loops (L1, L2, and L3) coordinating
the metal are shown schematically as gray ribbons. E256 from L3, which
forms an indirect bond via the equatorial water, has been removed
for clarity. The figure is rotated about a vertical axis by 180°
relative to B.
Emsley J, Knight CG, Farndale RW, Barnes MJ, Liddington RC. Structural basis of collagen recognition by integrin alpha2beta1. Cell. 2000 Mar 31;101(1):47-56. PubMed
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Talin-Integrin Complex
Structural determinants of integrin recognition by talin. The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior. |
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Figure:
Structural Studies of Talin-Integrin Binding(A) Ratio of 2D NMR 1H-15N
signal intensities of the full-length 3 tail, fused to a coiled-coil
construct, in the absence, I0, and presence, I, of F2+F3. Pronounced
reductions in peak intensity in the presence of F2+F3, i.e., low I/I0
ratios were observed for some residues, indicating a perturbation
of the associated amino acid residue by the interaction with F2+F3.
The absence of reductions of peak intensity within the coiled-coil
region (residues 3-39) confirms the specificity of the interactions.
The most strongly affected regions in the vicinity of the NPxY motif
are boxed, and the inset shows aligned sequences of integrins and
layilin (Lay1 and Lay2). The residue in position -8 of integrins (-6
in layilin) is in a blue box, while the NPXY motif is in a yellow
box. The observed effects are strongly dependent on the integrity
of the NPxY motif, as demonstrated by the abolition of these effects
by the Y747A substitution. The significance of the spectral changes
observed in the membrane proximal region is unclear.(B) Surface representation
of the F3 subdomain of talin, colored by electrostatic potential (blue
for positive charge and red for negative). The 3 integrin ligand,
residues 738-747, is shown as sticks. Integrin residues are labeled
in green, while residues of talin are labeled on the surface.(C) Stereo
close-up of the integrin-talin interaction as observed in the 3(739-749)-talin
chimera; the talin backbone is in gray, integrin in yellow. Residue
numbers are those for authentic talin (gray) and integrin (green)
sequences. For the latter, numbers in parenthesis refer to the position
with respect to the NPLY tyrosine. There are no significant differences
with 3(739-749)-talin. The main chain of one residue derived from
the construct upstream of W739 is also shown, as it has good electron
density in all of the chimeras. A partially buried water molecule
is indicated with (omega) and key H bonds with dotted lines.
Garcia-Alvarez
B, de Pereda JM, Calderwood DA, Ulmer TS, Critchley D, Campbell
ID, Ginsberg MH, Liddington RC. Structural determinants of integrin recognition by talin. Mol Cell. 2003 Jan;11(1):49-58. PubMed;
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FAT-FAK Structure
The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.
Focal adhesion kinase (FAK) is a tyrosine kinase found in focal
adhesions, intracellular signaling complexes that are formed following
engagement of the extracellular matrix by integrins. The C-terminal
'focal adhesion targeting' (FAT) region is necessary and sufficient
for localizing FAK to focal adhesions. We have determined the crystal
structure of FAT and show that it forms a four-helix bundle that
resembles those found in two other proteins involved in cell adhesion,
alpha-catenin and vinculin. The binding of FAT to the focal adhesion
protein, paxillin, requires the integrity of the helical bundle,
whereas binding to another focal adhesion protein, talin, does not.
We show by mutagenesis that paxillin binding involves two hydrophobic
patches on opposite faces of the bundle and propose a model in which
two LD motifs of paxillin adopt amphipathic helices that augment
the hydrophobic core of FAT, creating a six-helix bundle. |
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Figure:
Structural comparisons and surface properties of FAT. a, Comparison
with domains of apolipoprotein E (apoE), vinculin and alpha-catenin,
showing the similar four-helix bundle architecture. Helices are colored
from the N-terminus: red, yellow, green and blue. The FAT domain is
rotated by 90° about a vertical axis compared to Fig. 1b. The
vinculin domain contains a fifth helix at its N-terminus, shown as
a coil. b, c, Two views of the surface properties of FAT. The orientation
in (b) is related by a 180° rotation about a vertical axis. At
left, electrostatic potential surface of FAT, contoured from -15 (red)
to +15 (blue) kT e-1. The predicted location of paxillin LD motif
helices are shown as yellow coils. Residues mutated in this study
are indicated. At center are sequence conservation across species:
invariant (red), highly conserved (orange), conserved (yellow) and
variable (gray). At right, surface hydrophobicity of FAT. Hydrophobic
areas are shown in green; hydrophilic, magenta. Two prominent hydrophobic
patches (HP1 and HP2) implicated in paxillin binding are indicated.
Hayashi I, Vuori K, Liddington RC. The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nat Struct Biol. 2002 Feb;9(2):101-6. PubMed Other related PDB entries;
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Vinculin Structure
Crystal structure of the vinculin tail suggests a pathway for activation. Vinculin plays a dynamic role in the assembly of the actin cytoskeleton. A strong interaction between its head and tail domains that regulates binding to other cytoskeletal components is disrupted by acidic phospholipids. Here, we present the crystal structure of the vinculin tail, residues 879-1066. Five amphipathic helices form an antiparallel bundle that resembles exchangeable apolipoproteins. A C-terminal arm wraps across the base of the bundle and emerges as a hydrophobic hairpin surrounded by a collar of basic residues, adjacent to the N terminus. We show that the C-terminal arm is required for binding to acidic phospholipids but not to actin, and that binding either ligand induces conformational changes that may represent the first step in activation. |
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Figure:
Structure of full-length vinculin in its autoinhibited state. Domains
are shown in different colours: D1, residues 6-252; D2, 253-485; D3,
493-717; D4, 719-835; D5 (Vt), 896-1,066. A short N-terminal strand
('N', residues 1-5) precedes D1. The proline-rich region (838-878)
is partly disordered and precedes a 'strap' (residues 878-890), that
lies across the surface of Vt and the C terminus ('C'). Binding sites
for major ligands are indicated. PIP2, PtdIns(4,5)P2.
Bakolitsa C,
Cohen DM, Bankston LA, Bobkov AA, Cadwell GW, Jennings L, Critchley
DR, Craig SW, Liddington RC. Structural basis for vinculin activation at sites of cell adhesion. Nature. 2004 Jul 29;430(6999):583-6.
PubMed. |
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Structural basis for vinculin activation at sites of cell adhesion.
Vinculin is a highly conserved intracellular protein with a crucial
role in the maintenance and regulation of cell adhesion and migration.
In the cytosol, vinculin adopts a default autoinhibited conformation.
On recruitment to cell-cell and cell-matrix adherens-type junctions,
vinculin becomes activated and mediates various protein-protein
interactions that regulate the links between F-actin and the cadherin
and integrin families of cell-adhesion molecules. Here we describe
the crystal structure of the full-length vinculin molecule (1,066
amino acids), which shows a five-domain autoinhibited conformation
in which the carboxy-terminal tail domain is held pincer-like by
the vinculin head, and ligand binding is regulated both sterically
and allosterically. We show that conformational changes in the head,
tail and proline-rich domains are linked structurally and thermodynamically,
and propose a combinatorial pathway to activation that ensures that
vinculin is activated only at sites of cell adhesion when two or
more of its binding partners are brought into apposition. |
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Figure:
Stereo ribbon representation of Vt viewed from the side of the bundle.
Helices are shown in spectral colors from blue to red and labeled
H0-H5. The side chains of all aromatic residues are shown as ball-and-stick
and colored by atom type. The N-terminal arm shown is in the consensus
conformation found in the two molecules of the monoclinic crystal
form. The C terminus is that of molecule 1 in the orthorhombic form.
A consensus binding site for actin is contained within the first three
helices, H1-H3.
Bakolitsa C,
de Pereda JM, Bagshaw CR, Critchley DR, Liddington RC. Crystal structure of the vinculin tail suggests a pathway for activation. Cell. 1999
Dec 10;99(6):603-13. PubMed Other related PDB entries;
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Method Development
Quantitative fitting of atomic models into observed densities derived by electron microscopy. A new methodology for fitting atomic models into density distributions was developed. This approach is based on a global density correlation analysis that can be optionally supplemented by biochemical as well as biophysical data. The procedure is completely general and enables an objective evaluation of the resulting docking in the light of available biochemical and biophysical information as well as density correlation alone. We exhaustively tested the algorithms on calculated data and applied the approach successfully to several experimental data sets. Contact niels@burnham.org for program availability. Volkmann N, Hanein D. Quantitative fitting of atomic models into observed densities derived by electron microscopy. J Struct Biol. 1999 Apr-May;125(2-3):176-84 PubMed and Docking of atomic models into reconstructions from electron microscopy. Methods Enzymol. 2003;374:204-25. PubMed
A novel three-dimensional variant of the watershed transform for segmentation of electron density maps. Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratios. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. A segmentation approach targeting three-dimensional electron density maps derived by electron microscopy was developed. The approach consists of a novel three-dimensional variant of the immersion-based watershed algorithm. We tested the algorithm on calculated data and applied it to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures. The results indicate that the algorithm is reliable, efficient, accurate, and applicable to a wide variety of biological problems. Contact niels@burnham.org for program availability. |
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Figure:
Application of our 3D watershed algorithm to electron tomograms of
2D arrays of actin crosslinked by aldolase (data provided by J. Liu,
D. Taylor, and K.A. Taylor). (a) A portion of a raw tomogram. It is
clear that this type of density is difficult to interpret by eye.
(b) After the first step, a coarse-grained run of our segmentation
algorithm, connected areas that contain interesting features are emerging.
(c) One area is selected for further processing (white box in b).
(d) After a fine-grained segmentation of the selected volume, it becomes
possible to distinguish smaller entities within this area. Also, potential
artifacts or contaminations can be identified and eliminated. (e)
In the cleaned up map the actin filaments, the occasional individual
actin monomer within the filaments, and the crosslinking aldolase
(white box) can be readily identified and extracted. (f) Extracted
aldolase molecule. Note how well the shape and subdivision of the
extracted feature (left-hand side) correspond to the crystal structure
of aldolase (right-hand side).
Volkmann N. A novel three-dimensional variant of the watershed transform for segmentation of electron density maps. J Struct Biol. 2002 Apr-May;138(1-2):123-9. PubMed |
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Use of reduced representation templates for pattern recognition.
Reduced representation templates were used in a real-space pattern matching framework to facilitate automatic particle picking from electron micrographs. The procedure consists of five parts. First, reduced templates are constructed either from models or directly from the data. Second, a real-space pattern matching algorithm is applied using the reduced representations as templates. Third, peaks are selected from the resulting score map using peak-shape characteristics. Fourth, the surviving peaks are tested for distance constraints. Fifth, a correlation-based outlier screening is applied. Test applications to a data set of keyhole limpet hemocyanin particles indicate that the method is robust and reliable. Contact niels@burnham.org for program availability. Volkmann N. J Struct Biol. 2004 Jan-Feb;145(1-2):152-6. PubMed |
