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Focal adhesions (FAs) are large submembrane signaling complexes formed at sites of cellular attachment to the extracellular matrix. The interaction of LD motifs with their targets plays an important role in the assembly of FAs. We have determined the molecular basis for the recognition of two paxillin LD motifs by the FA targeting (FAT) domain of FA kinase using a combination of X-ray crystallography, solution NMR, and homology modeling. The four-helix FAT domain displays two LD binding sites on opposite sites of the molecule that bind LD peptides in a helical conformation. Threading studies suggest that the LD-interacting domain of p95PKL shares a common four-helical core with the FAT domain and the tail of vinculin, defining a structural family of LD motif binding modules.

The regulatory domains of Src family kinases SH3 and SH2 suppress Src activity when bound to the catalytic domain. Here, the isolated SH3-SH2 fragment from the Src family member Fyn (FynSH32) is studied by NMR. The properties of this fragment are expected to be similar to the domains in the active state, where they are dissociated from the catalytic domain. Crosscommunication between SH3 and SH2 of FynSH32, measured by chemical shift perturbation, was found to be small. Diffusion and alignment anisotropy measurements showed that SH3 and SH2 of peptide-bound FynSH32 are significantly coupled but still exhibit some interdomain flexibility. The observed average domain orientation indicates that a large SH3-SH2 domain closure is required to reach the inactive state. The implications of these results for Src regulation are discussed.

Background: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. Results: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. Conclusions: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

Background: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA–protein interactions remains poorly understood. Results: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA 4 –HA 16 ). All oligomers bound (except HA 4 ) with K d values ranging from 0.2–0.5 μM at 25°C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan–protein interactions. The HA 8 recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the β4/β5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. Conclusions: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

Summary Von Willebrand factor A (VWA) domains are versatile protein interaction domains with N and C termini in close proximity placing spatial constraints on overall protein structure. The 1.2 Å crystal structures of a collagen VI VWA domain and a disease-causing point mutant show C-terminal extensions that place the N and C termini at opposite ends. This allows a “beads-on-a-string” arrangement of multiple VWA domains as observed for ten N-terminal domains of the collagen VI α3 chain. The extension is linked to the core domain by a salt bridge and two hydrophobic patches. Comparison of the wild-type and a muscular dystrophy-associated mutant structure identifies a potential perturbation of a protein interaction interface and indeed, the secretion of mutant collagen VI tetramers is affected. Homology modeling is used to locate a number of disease-associated mutations and analyze their structural impact, which will allow mechanistic analysis of collagen-VI-associated muscular dystrophy phenotypes., Highlights • The structure of a VWA domain (N5) of collagen VI at 1.2 Å is presented • N and C termini of the domain are at opposite ends • The structure with a myopathy-causing mutation shows altered interaction interface • The impact of mutations in collagen VI VWA domains was analyzed, Becker et al. describe a crystal structure of a von Willebrand factor A (VWA) domain, which has a C-terminal extension that places the N- and C-termini at opposite ends not seen before for A domains. This allows a “beads-on-a-string” arrangement of multiple VWA domains as observed for the collagen VI α3 chain.