Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 Å resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a b-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin b-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.

BODIL is a molecular modeling environment geared to help the user to quickly identify key features of proteins critical to molecular recognition, especially (1) in drug discovery applications, and (2) to understand the structural basis for function. The program incorporates state-of-the-art graphics, sequence and structural alignment methods, among other capabilities needed in modern structure-function-drug target research. BODIL has a flexible design that allows on-the-fly incorporation of new modules, has intelligent memory management, and fast multi-view graphics. A beta version of BODIL and an accompanying tutorial are available at http://www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html.

Four integrins, namely ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 , form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their ␣ subunit. We have produced the human integrin ␣ 10 I domain as a recombinant protein to reveal its ligand binding specificity. In general, ␣ 10 I did recognize collagen types I-VI and laminin-1 in a Mg 2؉ -dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When ␣ 10 I was tested together with the ␣ 1 I and ␣ 2 I domains, all three I domains seemed to have their own collagen binding preferences. The integrin ␣ 2 I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin ␣ 1 I had the opposite binding pattern. The integrin ␣ 10 I domain was similar to the ␣ 1 I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the ␣ 1 I and ␣ 2 I domains, we modeled the structure of the ␣ 10 I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the ␣I domains, and their binding to types I, IV, and VI collagen was tested. In the ␣ 2 I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the ␣ 1 I and ␣ 10 I domains is oppositely charged (Arg-218). The mutation D219R in the ␣ 2 I domain changed the ligand binding pattern to resemble that of the ␣ 1 I and ␣ 10 I domains and, vice versa, the R218D mutation in the ␣ 1 I and ␣ 10 I domains created an ␣ 2 I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.Collagens are abundant structural proteins in the extracellular matrix. So far, 19 different triple helical protein trimers have been classified as a collagen subtype (1). The collagens can be grouped into subclasses according to their structural details. Many collagen subtypes (namely types I, II, III, V, and XI) have long continuous triple helices, and they can form large fibrils. In other collagens the triple helix has interruptions. Some collagens form networks (types IV, VIII, and X) or beaded filaments (type VI). Other collagen subclasses include fibrilassociated collagen with short interruptions in the triple helices (collagen types IX, XII, XIV, XVI, and XIX), anchoring fibril-forming collagen (type VII), and transmembrane collagen (types XIII and XVII). Collagen types XV and XVIII are found in association with basement membranes (the multiplexins; see Ref.2).The integrins form a large family of heterodimeric cell surface receptors involved in cell-extracellular matrix as well a...

The integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 , and ␣ 11 ␤ 1 are referred to as a collagen receptor subgroup of the integrin family. Recently, both ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O ‫؍‬ hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, ␣ 11 ␤ 1 , recognizes collagens using C2C12 cells transfected with ␣ 11 cDNA and the bacterially expressed recombinant ␣ 11 I domain. The ligand binding properties of ␣ 11 ␤ 1 were compared with those of ␣ 2 ␤ 1 . Mg 2؉ -dependent ␣ 11 ␤ 1 binding to type I collagen required micromolar Ca 2؉ but was inhibited by 1 mM Ca 2؉ , whereas ␣ 2 ␤ 1 -mediated binding was refractory to millimolar concentrations of Ca 2؉ . The bacterially expressed recombinant ␣ 11 I domain preference for fibrillar collagens over collagens IV and VI was the same as the ␣ 2 I domain. Despite the difference in Ca 2؉ sensitivity, ␣ 11 ␤ 1 -expressing cells and the ␣ 11 I domain bound to helical GFOGER sequences in a manner similar to ␣ 2 ␤ 1 -expressing cells and the ␣ 2 I domain. Modeling of the ␣ I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , and also ␣ 11 ␤ 1 integrins. Although ␣ 10 and ␣ 11 chains show the highest sequence identity, ␣ 2 and ␣ 11 are more similar with regard to collagen specificity. Future studies will reveal whether ␣ 2 ␤ 1 and ␣ 11 ␤ 1 integrins also show overlapping biological functions.The collagen family currently includes at least 24 members (1, 2), and four different collagen-binding integrins ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , ␣ 10 ␤ 1 (3) and ␣ 11 ␤ 1 (4) are known. The ␣ 3 ␤ 1 integrin does not interact directly with collagen, but it does act as a laminin receptor (5) that can affect the activity of the collagen receptor ␣ 2 ␤ 1 through receptor cross-talk (6).

Filamins (FLN) are large dimeric proteins that cross-link actin and work as important scaffolds in human cells. FLNs consist of an N-terminal actin-binding domain followed by 24 immunoglobulin-like domains (FLN1-24). FLN domains are divided into four subgroups based on their amino acid sequences. One of these subgroups, including domains 4, 9, 12, 17, 19, 21, and 23, shares a similar ligand-binding site between the β strands C and D. Several proteins, such as integrins β2 and β7, glycoprotein Ibα (GPIbα), and migfilin, have been shown to bind to this site. Here, we computationally estimated the binding free energies of filamin A (FLNa) subunits with bound peptides using the molecular mechanics-generalized Born surface area (MMGBSA) method. The obtained computational results correlated well with the experimental data, and they ranked efficiently both the binding of one ligand to all used FLNa-domains and the binding of all used ligands to FLNa21. Furthermore, the steered molecular dynamics (SMD) simulations pinpointed the binding hot spots for these complexes. These results demonstrate that molecular dynamics combined with free energy calculations are applicable to estimating the energetics of protein-protein interactions and can be used to direct the development of novel FLN function modulators.