Hideki Hatanaka

Solution Structure of the Link Module: A Hyaluronan-Binding Domain Involved in Extracellular Matrix Stability and Cell Migration

Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

SOLUTION STRUCTURE OF MIDKINE, A NEW HEPARIN-BINDING GROWTH FACTOR

Midkine (MK) is a 13 kDa heparin‐binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C‐terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel β‐strands, but the C‐terminal domain has a long flexible hairpin loop where a heparin‐binding consensus sequence is located. Basic residues on the β‐sheet of the C‐terminal domain form another heparin‐binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head‐to‐head dimer model of MK. The model was supported by the results of cross‐linking experiments using transglutaminase. The dimer has a fused heparin‐binding site at the dimer interface of the C‐terminal domain, and the heparin‐binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin‐binding activity and biological activity of the dimer.

Tertiary structure of destrin and structural similarity between two actin-regulating protein families.

Destrin is an isoprotein of cofilin that regulates actin cytoskeleton in various eukaryotes. We determined the tertiary structure of destrin by triple-resonance multidimensional nuclear magnetic resonance. In spite of there being no significant amino acid sequence homology, we found that the folding of destrin was strikingly similar to that of repeated segments in the gelsolin family, which resulted in a new protein fold group. Sequential dissimilarity of the actin-binding helix of destrin to that of gelsolin explains the Ca2+-independent actin-binding of destrin. Possible mechanisms of phosphorylation-sensitive phosphoinositide-competitive actin binding, of pH-dependent filament severing, and of nuclear translocation with actin in response to stresses, are discussed on the basis of the tertiary structure.

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed states suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Solution Structure of Der f 2, the Major Mite Allergen for Atopic Diseases

House dust mites cause heavy atopic diseases such as asthma and dermatitis. Among allergens from Dermatophagoides farinae, Der f 2 shows the highest positive rate for atopic patients, but its biological function in mites has been perfectly unknown, as well as the functions of its homologs in human and other animals. We have determined the tertiary structure of Der f 2 by multidimensional nuclear magnetic resonance spectroscopy. Der f 2 was found to be a single-domain protein of immunoglobulin fold, and its structure was the most similar to those of the two regulatory domains of transglutaminase. This fact, binding to the bacterial surface, and other small pieces of information hinted that Der f 2 is related to the innate antibacterial defense system in mites. The immunoglobulin E epitopes are also discussed on the basis of the tertiary structure.

Crucial Commitment of Proteolytic Activity of a Purified Recombinant Major House Dust Mite Allergen Der p1 to Sensitization toward IgE and IgG Responses

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-γ. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

Structure and ligand recognition of the PB1 domain: a novel protein module binding to the PC motif

PB1 domains are novel protein modules capable of binding to target proteins that contain PC motifs. We report here the NMR structure and ligand‐binding site of the PB1 domain of the cell polarity establishment protein, Bem1p. In addition, we identify the topology of the PC motif‐containing region of Cdc24p by NMR, another cell polarity establishment protein that interacts with Bem1p. The PC motif‐containing region is a structural domain offering a scaffold to the PC motif. The chemical shift perturbation experiment and the mutagenesis study show that the PC motif is a major structural element that binds to the PB1 domain. A structural database search reveals close similarity between the Bem1p PB1 domain and the c‐Raf1 Ras‐binding domain. However, these domains are functionally distinct from each other.

Solution structure and ligand-binding site of the carboxy-terminal SH3 domain of GRB2.

BACKGROUND Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein with three Src homology (SH) domains in the order SH3-SH2-SH3. Both SH3 domains of GRB2 are necessary for interaction with the protein Son of sevenless (Sos), which acts as a Ras activator. Thus, GRB2 mediates signal transduction from growth factor receptors to Ras and is thought to be a key molecule in signal transduction. RESULTS The three-dimensional structure of the carboxy-terminal SH3 domain of GRB2 (GRB2 C-SH3) was determined by NMR spectroscopy. The SH3 structure consists of six beta-strands arranged in two beta-sheets that are packed together perpendicularly with two additional beta-strands forming the third beta-sheet. GRB2 C-SH3 is very similar to SH3 domains from other proteins. The binding site of the ligand peptide (VPP-PVPPRRR) derived from the Sos protein was mapped on the GRB2 C-SH3 domain indirectly using 1H and 15N chemical shift changes, and directly using several intermolecular nuclear Overhauser effects. CONCLUSIONS Despite the structural similarity among the known SH3 domains, the sequence alignment and the secondary structure assignments differ. We therefore propose a standard description of the SH3 structures to facilitate comparison of individual SH3 domains, based on their three-dimensional structures. The binding site of the ligand peptide on GRB2 C-SH3 is in good agreement with those found in other SH3 domains.

Lipopolysaccharide binding of the mite allergen Der f 2

Lipid‐binding properties and/or involvement with host defense are often found in allergen proteins, implying that these intrinsic biological functions likely contribute to the allergenicity of allergens. The group 2 major mite allergens, Der f 2 and Der p 2, show structural homology with MD‐2, the lipopolysaccharide (LPS)‐binding component of the Toll‐like receptor (TLR) 4 signalling complex. Elucidation of the ligand‐binding properties of group 2 mite allergens and identification of interaction sites by structural studies are important to explore the relationship between allergenicity and biological function. Here, we report a ligand‐fishing approach in which His‐tagged Der f 2 was incubated with sonicated stable isotope‐labelled Escherichia coli as a potential ligand source, followed by isolation of Der f 2‐bound material by a HisTrap column and NMR analysis. We found that Der f 2 binds to LPS with a nanomolar affinity and, using fluorescence and gel filtration assays that LPS binds to Der f 2 in a molar ratio of 1 : 1. We mapped the LPS‐binding interface of Der f 2 by NMR perturbation studies, which suggested that LPS binds Der f 2 between the two large β‐sheets, similar to its binding to MD‐2, the LPS‐binding component of the innate immunity receptor TLR4.

Solution structure of human insulin-like growth factor II; recognition sites for receptors and binding proteins.

The three‐dimensional structure of human insulin‐like growth factor II was determined at high resolution in aqueous solution by NMR and simulated annealing based calculations. The structure is quite similar to those of insulin and insulin‐like growth factor I, which consists of an alpha‐helix followed by a turn and a strand in the B‐region and two antiparallel alpha‐helices in the A‐region. However, the regions of Ala1‐Glu6, Pro31‐Arg40 and Thr62‐Glu67 are not well‐defined for lack of distance constraints, possibly due to motional flexibility. Based on the resultant structure and the results of structure‐activity relationships, we propose the interaction sites of insulin‐like growth factor II with the type 2 insulin‐like growth factor receptor and the insulin‐like growth factor binding proteins. These sites partially overlap with each other at the opposite side of the putative binding surface to the insulin receptor and the type 1 insulin‐like growth factor receptor. We also discuss the interaction modes of insulin‐like growth factor II with the insulin receptor and the type 1 insulin‐like growth factor receptor.