Journal of Molecular Biology
Structure, Evolutionary Conservation, and Conformational Dynamics of Homo sapiens Fascin-1, an F-actin Crosslinking Protein
Introduction
The actin cytoskeleton of eukaryotic cells is centrally involved in a range of cellular functions including migration, endocytosis and division. In each case, the cell uses a host of accessory proteins to regulate actin dynamics spatiotemporally to achieve cellular function. In the case of migration, the leading edge of the cell consists predominantly of two types of protrusive filamentous actin (F-actin) structures: the dendritic sheetlike lamellipodium1, 2, 3, 4, 5 and dynamic cortical spike-like filopodia.6, 7, 8 Filopodia consist of compact ordered bundles of unipolar actin filaments that are nucleated at the leading edge by formins9, 10 and crosslinked tightly into highly ordered bundles by the actin-binding protein fascin, which is both highly conserved evolutionarily and tissue- and cell-type-specific.7, 11 While fascin dominates in cortical actin bundles such as filopodia, oocyte microvilli, and the dendrites of dendritic cells, which play direct roles in cell migration, cell-matrix adhesion, and cell-cell interactions, fascin is also associated to a lesser extent with cytoplasmic actin bundles that participate in the maintenance of internal cell architecture.12 Fascin is additionally known to be upregulated in a number of highly motile cell phenotypes including invasive cancer cells.13, 14, 15, 16, 17
It is well established that the molecular size and conformational flexibility of actin crosslinking proteins are highly correlated with the morphological cytoskeletal structures with which they are associated.18, 19, 20, 21 For example, human filamin is a relatively large homodimer (approximately 160 nm molecular dimension) consisting of 24 tandem immunoglobulin repeats that is associated primarily with dendritic cytoskeletal networks,22 α-actinin is a smaller (35 nm) anti-parallel homodimer that is associated with both networks and bundles such as the contractile ring in dividing cells and stress fibers in adherent cells,23, 24 and the compact ABP fimbrin (10 nm)25 (also called plastin) is found nearly exclusively in highly ordered unipolar actin bundles such as microvilli. The X-ray structures of several of these and related actin-crosslinking proteins have been determined. They consist of dual calponin homology actin-binding domains that are suggested to stabilize actin filaments.26, 27, 28, 29, 30, 31, 32 Fimbrin additionally has two N-terminal calcium-binding EF-hand motifs that confer calcium regulation of its F-actin crosslinking activity in human isoforms.33, 34
Homo sapiens fascin-1 also crosslinks actin filaments into compact unipolar bundles, but in a manner that is regulated by phosphorylation of serine 39 by protein kinase C,35 which inhibits its actin-bundling activity without affecting its localization to the filopodial tip complex.7 H. sapiens fascin-1 is a compact, 55 kDa (493 residues) globular monomer with putative actin-binding domains that differ in primary sequence. Fascin was originally discovered in extracts from sea urchin eggs36 and was later found in other invertebrates as well as vertebrates including Drosophilia,37 starfish sperm,38 Xenopus laevis,39 rodents40 and humans.41 H. sapiens fascin-1 is the original vertebrate fascin discovered, whereas retinal and testis fascin were discovered later and named fascin-2 and fascin-3, respectively.11 Sequence alignment shows no similarity between fascins and other known actin-binding proteins in humans, but strong similarities within the fascin family itself.42 Atomic models for actin-fascin bundles have been proposed on the basis of optical diffraction studies of negatively stained reconstituted material.43 These suggest an 11 nm transverse banding pattern and uniformly polarized actin filaments organized in a hexagonal array with an interfilament distance of 11.5 nm. Assuming a single fascin monomer per crosslink, the predicted fascin-actin stoichiometry of 1:4.5 is in good agreement with experimentally measured ratios.44 This maximal stoichiometry is a result of the helical twist of the actin filament that limits crosslinking sites between pairs of filaments to every fourth or fifth actin monomer. Fascin is additionally suggested to impart unusual mechanical stiffness to actin bundles in cells7,45, 46, 47 and reconstituted actin systems24, 48, 49 as compared with fimbrin, despite similar actin-binding affinities and bundle structure.43, 50 Despite its importance to cell function, the molecular basis for the unique localization and actin-bundling properties of fascin are not known. Here, we report the 2.9 Å resolution crystal structure of full-length recombinant H. sapiens fascin-1 and examine its packing, evolutionary sequence conservation and conformational flexibility. The results of normal mode analysis (NMA) of fascin suggest potential functional consequences of its distinct molecular structure and flexibility in crosslinking F-actin. Mutational and other experimental studies are needed to test these predictions.
Section snippets
Overall structure
The two fascin molecules in the asymmetric unit are highly similar, with a root-mean-square deviation (RMSD) of 0.64 Å for 474 Cα atom pair equivalences. Fascin is composed of four tandem repeat β-trefoil domains (Fig. 1) with pseudo 3-fold symmetry, consisting of 12 β-strands that form the barrel (B) and cap (C) regions in the sequence: BCCBBCCBBCCB (Fig. 2a). The domains pack to form a distorted tetrahedron composed of two lobes: domains F1 (residues 8–139) and F2 (residues 140–260) form the
Discussion
The crystal structure of H. sapiens fascin-1 reveals that its β-trefoil domains associate via internal hydrophobic interactions and external ionic interactions at their bases to form cylindrical β-barrel lobes. NMA indicates that these interactions confer structural integrity to each cylindrical lobe that is maintained in the full-length molecule. Conservation analysis confirms the functional importance of the cores of the β-trefoil domains, which contain bulky hydrophobic residues that are
Purification and crystallization
Recombinant H. sapiens fascin was prepared using a procedure similar to that described earlier.35 H. sapiens fascin was expressed as a glutathione-S-transferase fusion using pGEX2T in the Escherichia coli strain JR600. Cultures were grown at 37 °C to an absorbance of 0.6 at 600 nm and cooled to 25 °C. Protein expression was induced by the addition of IPTG (final concentration 1 mM) and growth continued overnight at 25 °C. Cells were harvested by centrifugation, washed with PBS, resuspended in
Protein Data Bank accession numbers
X-ray coordinates and structure factors for H. sapiens fascin-1 have been deposited in the Protein Data Bank under accession code 1DFC.
Acknowledgements
The authors are grateful to Martin Karplus and Danijela Vignjevic for extensive comments on the manuscript. Funding from the Samuel A. Goldblith Professorship to M.B. and from the NIH to S.C.A. is gratefully acknowledged.
References (103)
- et al.
The lamellipodium: where motility begins
Trends Cell Biol.
(2002) - et al.
Cellular motility driven by assembly and disassembly of actin filaments
Cell
(2003) Actin crosslinking proteins at the leading edge
Semin. Cell Biol.
(1994)- et al.
Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis
Cell
(2004) Roles of fascin in cell adhesion and motility
Curr. Opin. Cell Biol.
(2004)- et al.
Roles of fascin in human carcinoma motility and signaling: prospects for a novel biomarker?
Int. J. Biochem. Cell Biol.
(2005) - et al.
Fascin, an actin-bundling protein, modulates colonic epithelial cell invasiveness and differentiation in vitro
Am. J. Pathol.
(2003) Parallel actin bundles and their multiple actin-bundling proteins
Curr. Opin. Cell Biol.
(2000)- et al.
A 3-D reconstruction of smooth muscle alpha-actinin by CryoEM reveals two different conformations at the actin-binding region
J. Mol. Biol.
(2004) - et al.
Micromechanics and ultrastructure of actin filament networks crosslinked by human fascin: A comparison with alpha-actinin
J. Mol. Biol.
(2001)
Structure of the actin crosslinking core of fimbrin
Structure
The 2.0 Å structure of the second calponin homology domain from the actin-binding region of the dystrophin homologue utrophin
J. Mol. Biol.
Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer
Struct. Fold. Des.
Structure of the alpha-actinin rod: Molecular basis for cross-linking of actin filaments
Cell
Identification of an actin binding region and a protein kinase C phosphorylation site on human fascin
J. Biol. Chem.
A vertebrate homolog of the actin-bundling protein fascin
Biochim. Biophys. Acta Gene Struct. Expression
Cloning and expression of a murine fascin homolog from mouse brain
J. Biol. Chem.
Separation and interaction of major components of sea urchin actin gel
J. Mol. Biol.
Cytoskeletal bundle mechanics
Biophys. J.
Morphogenesis of liposomes encapsulating actin depends on the type of actin-crosslinking
J. Mol. Biol.
Beta-trefoil fold patterns of structure and sequence in the Kunitz inhibitors interleukins-1-beta and 1-alpha and fibroblast growth factors
J. Mol. Biol.
The Protein Data Bank: a computer-based archival file for macromolecular structures
J. Mol. Biol.
SCOP: A structural classification of proteins database for the investigation of sequences and structures
J. Mol. Biol.
Crystal structure of recombinant human interleukin-1β at 2.0 Å resolution
J. Mol. Biol.
Identification of distant homologues of fibroblast growth factors suggests a common ancestor for all beta-trefoil proteins
J. Mol. Biol.
Three-fold structural pattern in the soybean trypsin-inhibitor (Kunitz)
J. Mol. Biol.
Crystal structure of abrin-A at 2.14 Å
J. Mol. Biol.
Crystal-structure of a Kunitz-type trypsin-inhibitor from Erythrina caffra seeds
J. Mol. Biol.
Hisactophilin, a histidine-rich actin-binding protein from Dictyostelium discoideum
J. Biol. Chem.
Phosphorylation of human fascin inhibits its actin binding and bundling activities
J. Biol. Chem.
Interactive computer graphics: FRODO
Methods Enzymol.
ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information
J. Mol. Biol.
A family of evolution-entropy hybrid methods for ranking protein residues by importance
J. Mol. Biol.
An evolutionary trace method defines binding surfaces common to protein families
J. Mol. Biol.
VMD: visual molecular dynamics
J. Mol. Graph.
Salt bridge stability in monomeric proteins
J. Mol. Biol.
A coarse-grained normal mode approach for macromolecules: An efficient implementation and application to Ca(2+)-ATPase
Biophys. J.
A simple method for displaying the hydropathic character of a protein
J. Mol. Biol.
The Croonian Lecture, 1978: The crawling movement of metazoan cells
Proc. Roy. Soc. B
Mechanism of actin-based motility
Science
The making of filopodia
Curr. Opin. Cell Biol.
Role of fascin in filopodial protrusion
J. Cell Biol.
Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction
J. Cell Biol.
Mechanism and function of formins in the control of actin assembly
Annu. Rev. Biochem.
Fascins, and their roles in cell structure and function
BioEssays
Fascin, a novel target of beta-Catenin-TCF signaling, is expressed at the invasive front of human colon cancer
Cancer Res.
Expression of fascin, an actin-bundling protein, in astrocytomas of varying grades
Brain Tumor Pathol.
Dual actin-bundling and protein kinase c-binding activities of fascin regulate carcinoma cell migration downstream of Rac and contribute to metastasis
Mol. Biol. Cell
Modular organization of actin cross-linking proteins
Trends Biochem. Sci.
The co-workers of actin filaments: From cell structures to signals
Nat. Rev. Mol. Cell Biol.
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