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Junichi Takagi

Publications and source records attributed to Junichi Takagi.

33 records · Page 2Linked to original sources
PubMed2003

High affinity ligand binding by integrins does not involve head separation.

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers alpha- and beta-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the alpha-subunit beta-propeller domain and the beta-subunit I-like domain. Basal and activation-dependent ligand binding by alpha(IIb)beta(3) and alpha(V)beta(3) was unaffected. The covalent linkage prevented dissociation of alpha(IIb)beta(3) into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type alpha(IIb)beta(3) on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between alpha(IIb) and beta(3) headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated alphabeta headpiece.

Animals↗
PubMed2003

Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation.

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.

Amino Acid Substitution↗
PubMed2002

Stabilizing the integrin alpha M inserted domain in alternative conformations with a range of engineered disulfide bonds.

Conformational movement of the C-terminal alpha7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an alpha/beta Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the alpha7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cbeta atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active alphaMbeta2 heterodimers and isolated alphaM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive alphaMbeta2 that was resistant to activation but became activatable after dithiothreitol treatment.

Dimerization↗
PubMed2002

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling.

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.

Humans↗
PubMed2002

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding.

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.

Amino Acid Sequence↗
PubMed2002

Fibronectin peptides derived from two distinct regions stimulate adipocyte differentiation by preventing fibronectin matrix assembly.

Here, we show that fibronectin (FN) peptides derived from two distinct regions promote the insulin-induced adipocyte differentiation of ST-13 cells by preventing FN fibrillogenesis. ST-13 cells formed numerous FN fibrils under nonadipogenic conditions, whereas this FN fibrillogenesis was suppressed by adipose induction with insulin. The insulin-induced adipocyte differentiation was promoted by an amino-terminal 24-kDa fragment of FN, accompanied by further suppression of FN fibrillogenesis. The 24 K fragment prevented FN matrix assembly by direct incorporation into the FN matrix. Like the 24 K fragment, a peptide from the 14th type III repeat, termed FNIII14, which suppressed the integrin alpha 5 beta 1-mediated adhesion of ST-13 cells to FN, accelerated the adipocyte differentiation by preventing FN fibrillogenesis without direct incorporation into the FN matrix. FNIII14 induced the conformation change of beta1 integrins of K562 cells from active to resting, as judged by FACS analysis using a monoclonal antibody AG89 directed to an active beta1 integrin-dependent epitope. Binding of a (125)I-labeled FN fragment containing the RGD cell adhesive site to ST-13 cell surface was dissociated by FNIII14, with a concomitant binding of FNIII14 itself to the cell surface. The affinity labeling of ST-13 cells using biotinylated FNIII14 showed that FNIII14 specifically bound to a nonintegrin membrane protein with M(r) of around 50 kDa. Thus, the results indicated that prevention of FN fibrillogenesis by the 24 K Fib 1 fragment and FNIII14 caused the promotion of adipocyte differentiation of ST-13 cells and that the former was due to the direct incorporation into the FN matrix and that the latter might be interpreted by negative regulation of FN receptor alpha 5 beta 1 activity.

Adipocytes↗
PubMed2002

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides.

Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and N(alpha)-methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.

Amino Acid Sequence↗
PubMed2002

Archaeal surface layer proteins contain beta propeller, PKD, and beta helix domains and are related to metazoan cell surface proteins.

The surface layer of archaeobacteria protects cells from extreme environments and, in Methanosarcina, may regulate cell adhesion. We identify three domain types that account for the complete architecture of numerous Methanosarcina surface layer proteins (SLPs). We solve the crystal structure for two of these domains, which correspond to the two N-terminal domains of an M. mazei SLP. One domain displays a unique, highly symmetrical, seven-bladed beta propeller fold, and the other belongs to the polycystic kidney disease (PKD) superfamily fold. The third domain is predicted to adopt a beta helix fold. These domains have homologs in metazoan cell surface proteins, suggesting remarkable relationships between domains in archaeal SLPs and metazoan cell surface proteins.

Amino Acid Sequence↗
PubMed2002

Integrin activation and structural rearrangement.

Among adhesion receptor families, integrins are particularly important in biological processes that require rapid modulation of adhesion and de-adhesion. Activation on a timescale of < 1 s of beta2 integrins on leukocytes and beta3 integrins on platelets enables deposition of these cells at sites of inflammation or vessel wall injury. Recent crystal, nuclear magnetic resonance (NMR), and electron microscope (EM) structures of integrins and their domains lead to a unifying mechanism of activation for both integrins that contain and those that lack an inserted (I) domain. The I domain adopts two alternative conformations, termed open and closed. In striking similarity to signaling G-proteins, rearrangement of a Mg2+-binding site is linked to large conformational movements in distant backbone regions. Mutations that stabilize a particular conformation show that the open conformation has high affinity for ligand, whereas the closed conformation has low affinity. Movement of the C-terminal alpha-helix 10 A down the side of the domain in the open conformation is sufficient to increase affinity at the distal ligand-binding site 9,000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. Recent structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the integrin headpiece, and a critical role for integrin epidermal growth factor (EGF) domains in the stalk region. The headpiece of the integrin faces down towards the membrane in the inactive conformation, and extends upward in a "switchblade"-like opening upon activation. These long-range structural rearrangements of the entire integrin molecule involving interdomain contacts appear closely linked to conformational changes within the I and I-like domains, which result in increased affinity and competence for ligand binding.

Animals↗
PubMed2002

Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation.

Cysteine-rich repeats in the integrin beta subunit stalk region relay activation signals to the ligand-binding headpiece. The NMR solution structure and disulfide bond connectivity of Cys-rich module-3 of the integrin beta2 subunit reveal a nosecone-shaped variant of the EGF fold, termed an integrin-EGF (I-EGF) domain. Interdomain contacts between I-EGF domains 2 and 3 observed by NMR support a model in which the modules are related by an approximate two-fold screw axis in an extended arrangement. Our findings complement a 3.1 A crystal structure of the extracellular portion of integrin alphaVbeta3, which lacks an atomic model for I-EGF2 and a portion of I-EGF3. The disulfide connectivity of I-EGF3 chemically assigned here differs from the pairings suggested in the alphaVbeta3 structure. Epitopes that become exposed upon integrin activation and residues that restrain activation are defined in beta2 I-EGF domains 2 and 3. Superposition on the alphaVbeta3 structure reveals that they are buried. This observation suggests that the highly bent alphaVbeta3 structure represents the inactive conformation and that release of contacts with I-EGF modules 2 and 3 triggers a switchblade-like opening motion extending the integrin into its active conformation.

Amino Acid Motifs↗
PubMed2001

Conformational regulation of integrin structure and function.

Integrins are a structurally elaborate family of heterodimers that mediate divalent cation-dependent cell adhesion in a wide range of biological contexts. The inserted (I) domain binds ligand in the subset of integrins in which it is present. Its structure has been determined in two alternative conformations, termed open and closed. In striking similarity to signaling G proteins, rearrangement of a Mg(2+)-binding site is linked to large conformational movements in distant backbone regions. Mutations have been used to stabilize either the closed or open structures. These show that the snapshots of the open conformation seen only in the presence of a ligand or a ligand mimetic represent a high-affinity, ligand-binding conformation, whereas those of the closed conformation correspond to a low-affinity conformation. The C-terminal alpha-helix moves 10 A down the side of the domain in the open conformation. Locking in the conformation of the preceding loop is sufficient to increase affinity for ligand 9000-fold. This C-terminal "bell-rope" provides a mechanism for linkage to conformational movements in other domains. The transition from the closed to open conformation has been implicated in fast (<1 s) regulation of integrin affinity in response to activation signals from inside the cell. Recent integrin structures and functional studies reveal interactions between beta-propeller, I, and I-like domains in the headpiece, and a critical role for integrin EGF domains in the stalk region. These studies suggest that the headpiece of the integrin faces down toward the membrane in the inactive conformation and extends upward in a "switchblade"-like opening motion upon activation. These long-range structural rearrangements of the entire integrin molecule involving multiple interdomain contacts appear closely linked to conformational changes in the I domain, which result in increased affinity and competence for ligand binding.

Amino Acid Sequence↗