Assembly of a Filamin Four-domain Fragment and the Influence of Splicing Variant-1 on the Structure

Ulla Pentikäinen,Pengju Jiang,Heikki Takala,Salla Ruskamo,Iain D Campbell,Jari Ylänne

doi:10.1074/jbc.m110.195958

Ulla Pentikäinen, Pengju Jiang + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.195958

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Abstract

Filamins are scaffold proteins that bind to various proteins, including the actin cytoskeleton, integrin adhesion receptors, and adaptor proteins such as migfilin. Alternative splicing of filamin, largely constructed from 24 Ig-like domains, is thought to have a role in regulating its interactions with other proteins. The filamin A splice variant-1 (FLNa var-1) lacks 41 amino acids, including the last β-strand of domain 19, FLNa(19), and the first β-strand of FLNa(20) that was previously shown to mask a key binding site on FLNa(21). Here, we present a structural characterization of domains 18-21, FLNa(18-21), in the FLNa var-1 as well as its nonspliced counterpart. A model of nonspliced FLNa(18-21), obtained from small angle x-ray scattering data, shows that these four domains form an L-shaped structure, with one arm composed of a pair of domains. NMR spectroscopy reveals that in the splice variant, FLNa(19) is unstructured whereas the other domains retain the same fold as in their canonical counterparts. The maximum dimensions predicted by small angle x-ray scattering data are increased upon migfilin binding in the FLNa(18-21) but not in the splice variant, suggesting that migfilin binding is able to displace the masking β-strand and cause a rearrangement of the structure. Possible function roles for the spliced variants are discussed.

Highlights

The filamin family has three members; filamins (FLNs)4 A, B and C
NMR Reveals that FLNa(19) Is Well Folded in FLNa(18 –21) but Is Intrinsically Unfolded in FLNa Var-1—The solution behavior of FLNa(18 –21) and FLNa(18 –21) var-1 fragments was first studied by NMR
The NMR heteronuclear single quantum correlation spectroscopy (HSQC) spectra of domain pairs and the four-domain 18 –21 fragment are very similar (Fig. 2), whereas many peaks are missing in the splice variant, even when the 41 deleted residues are taken into account

Summary

EXPERIMENTAL PROCEDURES

Recombinant Proteins—FLNa fragments were amplified by PCR and cloned into modified pGEX vector (GE Healthcare) with a tobacco etch virus protease cleavage site. The ␤7 integrin (776PLYKSAITTTINP788)-derived peptide for NMR-based binding studies was the non-isotope-labeled Pro776–Pro788 as described previously (21). All NMR samples were buffered with 50 mM sodium phosphate (pH 6.10 or 7.00) containing 100 mM NaCl, 5 mM DTT (only for proteins with cysteine), and 0.02% sodium azide in 90% H2O and 10% D2O. N-terminal Sequencing—For N-terminal sequencing the protein fragments were run on 12.5% SDS-PAGE (23), electroblotted onto a polyvinylidene difluoride (PVDF) membrane (24) and stained with Coomassie Brilliant Blue. Proteins were eluted with 10 ␮l of SDSelectrophoresis sample buffer and run on a SDS-PAGE. Multiple runs of EOM were performed, and the results were averaged to provide quantitative information about the flexibility of the protein in solution (in particular, the Rg and Dmax distributions in the selected ensembles).

RESULTS

FLN isoform

DISCUSSION