Mode of substrate recognition by the Josephin domain of ataxin-3, which has an endo-type deubiquitinase activity.

Ataxin-3, which is encoded by a gene that has been associated with Machado-Joseph disease, contains a catalytic N-terminal Josephin domain with deubiquitinase activity. Here, we show that the Josephin domain of ataxin 3 catalyzes endo-type cleavage of Lys48-linked polyubiquitin. Furthermore, NMR data obtained following site-specific paramagnetic spin labeling of Lys48-linked di-ubiquitin revealed that both ubiquitin units interact with the Josephin domain, with the C-terminal Gly76 of the proximal unit being situated in the vicinity of the catalytic triad of Josephin domain.

NMR characterization of the interaction between the PUB domain of peptide:N-glycanase and ubiquitin-like domain of HR23.

PUB domains are identified in several proteins functioning in the ubiquitin (Ub)-proteasome system and considered as p97-binding modules. To address the further functional roles of these domains, we herein characterized the interactions of the PUB domain of peptide:N-glycanase (PNGase) with Ub and Ub-like domain (UBL) of the proteasome shuttle factor HR23. NMR data indicated that PNGase-PUB exerts an acceptor preferentially for HR23-UBL, electrostatically interacting with the UBL surface employed for binding to other Ub/UBL motifs.

Backbone and side chain 1H, 13C, and 15N assignments of the ubiquitin-like domain of human HOIL-1L, an essential component of linear ubiquitin chain assembly complex.

HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date.

Solution structure and dynamics of mouse ARMET.

ARMET is an endoplasmic reticulum (ER) stress-inducible protein that is required for maintaining cell viability under ER stress conditions. However, the exact molecular mechanisms by which ARMET protects cells are unknown. Here, we have analyzed the solution structure of ARMET.

NMR characterization of intramolecular interaction of osteopontin, an intrinsically disordered protein with cryptic integrin-binding motifs.

Osteopontin (OPN) is an integrin-binding protein found in a variety of tissues and physiological fluids and is involved in divergent biological processes such as migration, adhesion and signaling in integrin-independent as well as dependent manners. The adhesive activity of this protein is modulated upon cleavage by thrombin at the central part of the molecule, in the vicinity of the integrin-binding sequences. Although detailed structural characterization is crucial for further understanding of the regulatory mechanisms of the OPN functions, its intrinsically disordered property hampers in-depth conformational analyses.

Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency.

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses.

Redox-dependent domain rearrangement of protein disulfide isomerase coupled with exposure of its substrate-binding hydrophobic surface.

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b', and a', along with a C-terminal extension. The homologous a and a' domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b' domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides.

Characterization of inhibitor-bound alpha-synuclein dimer: role of alpha-synuclein N-terminal region in dimerization and inhibitor binding.

alpha-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other alpha-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit alpha-synuclein assembly with low micromolar IC(50) values, and that SDS-stable, noncytotoxic soluble alpha-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound alpha-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism.

Up-and-down topological mode of amyloid beta-peptide lying on hydrophilic/hydrophobic interface of ganglioside clusters.

Growing evidence has indicated that GM1 ganglioside specifically interacts with Amyloid beta-peptide (Abeta) and thereby promotes Alzheimer's disease-associated Abeta assembly. To characterize the conformation of Abeta bound to the ganglioside, we performed 920 MHz ultra-high field NMR analyses using isotopically labeled Abeta(1-40) in association with GM1 and lyso-GM1 micelles. Our NMR data revealed that (1) Abeta(1-40) forms discontinuous alpha-helices at the segments His(14)-Val(24) and Ile(31)-Val(36) upon binding to the gangliosidic micelles, leaving the remaining regions disordered, and (2) Abeta(1-40) lies on hydrophobic/hydrophilic interface of the ganglioside cluster exhibiting an up-and-down topological mode in which the two alpha-helices and the C-terminal dipeptide segment are in contact with the hydrophobic interior, whereas the remaining regions are exposed to the aqueous environment.

920 MHz ultra-high field NMR approaches to structural glycobiology.

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein.

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies.

Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1.

Removal of the fucose residue from the oligosaccharides attached to Asn297 of human immunoglobulin G1 (IgG1) results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to Fcgamma receptor IIIa. To provide structural insight into the mechanisms of affinity enhancement, we determined the crystal structure of the nonfucosylated Fc fragment and compared it with that of fucosylated Fc. The overall conformations of the fucosylated and nonfucosylated Fc fragments were similar except for hydration mode around Tyr296.

Solution structure and dynamics of Ufm1, a ubiquitin-fold modifier 1.

The ubiquitin-fold modifier 1 (Ufm1) is one of various ubiquitin-like modifiers and conjugates to target proteins in cells through Uba5 (E1) and Ufc1 (E2). The Ufm1-system is conserved in metazoa and plants, suggesting its potential roles in various multicellular organisms. Herein, we analyzed the solution structure and dynamics of human Ufm1 (hsUfm1) by nuclear magnetic resonance spectroscopy.

Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.

The Fc portion of immunoglobulin G (IgG) expresses the biantennary complex type oligosaccharides at Asn297 of the C(H)2 domain of each heavy chain with microheterogeneities depending on physiological and pathological states. These N-glycans are known to be essential for promotion of proper effector functions of IgG such as complement activation and Fcgamma receptor (FcgammaR)-mediated activities. To gain a better understanding of the role of Fc glycosylation, we prepared a series of truncated glycoforms of human IgG1-Fc and analyzed their interactions with human soluble FcgammaRIIIa (sFcgammaRIIIa) and with staphylococcal protein A by surface plasmon resonance and nuclear magnetic resonance (NMR) methods.

Terahertz time-domain spectroscopy of amino acids and polypeptides.

Frequency-dependent absorption coefficients and refractive indices of amino acids (glycine and l-alanine) and polypeptides (polyglycine and poly-l-alanine) in the wavenumber region from 7 to 55 cm(-1) were measured by terahertz time-domain spectroscopy. A vibrational band was observed at 45.5 cm(-1) for polyglycine, which was assigned as an interchain mode.

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification.

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel.

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone.

Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin.