Abstract

Filamins are large dimeric proteins that cross-link actin into three-dimensional bundles or orthogonal networks. In addition to an actin-binding domain, each filamin monomer contains 24 immunoglobulin-like domains separated by flexible regions between domains 15–16 and 23–24. Dimerisation of filamin occurs through the Ig-like domain 24. Filamins bind to a variety of molecules. They provide a link between the plasma membrane and the cytoskeleton through interactions with transmembrane receptors, and at the same time, serve as a platform for signalling molecules. Filamins are involved in several human diseases affecting the central nervous system, vascular system and muscle. In this study the structure of the the carboxy-terminus of filamin was resolved and details of filamins interaction with a platelet surface protein important in haemostasis were analysed.

An x-ray structure of the Ig-like domain 24 of human filamin C was solved at the resolution of 1.43 Å. The asymmetric unit of the crystal contains one monomer; a crystallographic dimer is formed by 2-fold axis symmetry. Point mutation studies confirmed that the dimer seen in the crystal is also present in solution. The structure showed that the dimerisation mode of human filamin is completely different from that in the Dictyostelium discoideum amoeba filamin analogue. Human filamin dimerises through β-strands C and D, and the Dictyostelium protein through β-strands B and G located on the opposite edge of the β-sandwich. Based on the sequence homology between vertebrate filamins it was proposed that the interface seen in human filamin is common for all vertebrate filamins.

The structure of human filamin C Ig-like domains 23–24 was solved by combining the techniques of x-ray crystallography and small angle x-ray scattering (SAXS). This structure provides further insight into the organization of the domains in the carboxy-terminal part of filamin molecule.

One of the first structural examples of the interaction of filamin with a ligand was provided by this study. The x-ray structure of filamin A domain 17 in complex with the alpha subunit of the GPIb-V-IX receptor was solved at a resolution of 2.3 Å. The interaction between filamin and the GPIbα-V-IX receptor is important for maintaining the integrity and shape of blood platelets, as well as for regulating the receptor adhesive function. This study also revealed that the Ig-like domain 17 represents a major binding site of filamin to GPIbα. The K_d of the interaction, determined by calorimetric studies, was 11 μM. The specificity of the filamin A 17 – GPIbα interaction is mainly determined by hydrophobic contacts.