Structure, Evolutionary Conservation, and Conformational Dynamics of Homo sapiens Fascin-1, an F-actin Crosslinking Protein

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Abstract

Eukaryotes have several highly conserved actin-binding proteins that crosslink filamentous actin into compact ordered bundles present in distinct cytoskeletal processes, including microvilli, stereocilia and filopodia. Fascin is an actin-binding protein that is present predominantly in filopodia, which are believed to play a central role in normal and aberrant cell migration. An important outstanding question regards the molecular basis for the unique localization and functional properties of fascin compared with other actin crosslinking proteins. Here, we present the crystal structure of full-length Homo sapiens fascin-1, and examine its packing, conformational flexibility, and evolutionary sequence conservation. The structure reveals a novel arrangement of four tandem β-trefoil domains that form a bi-lobed structure with approximate pseudo 2-fold symmetry. Each lobe has internal approximate pseudo 2-fold and pseudo 3-fold symmetry axes that are approximately perpendicular, with β-hairpin triplets located symmetrically on opposite sides of each lobe that mutational data suggest are actin-binding domains. Sequence conservation analysis confirms the importance of hydrophobic core residues that stabilize the β-trefoil fold, as well as interfacial residues that are likely to stabilize the overall fascin molecule. Sequence conservation also indicates highly conserved surface patches near the putative actin-binding domains of fascin, which conformational dynamics analysis suggests to be coupled via an allosteric mechanism that might have important functional implications for F-actin crosslinking by fascin.

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.

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