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Noritaka Nishida

Publications and source records attributed to Noritaka Nishida.

5 recordsLinked to original sources
PubMed2006

Structural transitions of complement component C3 and its activation products.

Complement sensitizes pathogens for phagocytosis and lysis. We use electron microscopy to examine the structural transitions in the activation of the pivotal protein in the complement pathway, C3. In the cleavage product C3b, the position of the thioester domain moves approximately 100 Angstrom, which becomes covalently coupled to antigenic surfaces. In the iC3b fragment, cleavage in an intervening domain creates a long flexible linker between the thioester domain and the macroglobulin domain ring of C3. Studies on two products of nucleophile addition to C3 reveal a structural intermediate in activation, and a final product, in which the anaphylatoxin domain has undergone a remarkable movement through the macroglobulin ring.

Chromatography, Gel↗
PubMed2006

Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy.

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the (13)C resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that (13)C NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.

Adenosine Diphosphate↗
PubMed2006

Activation of leukocyte beta2 integrins by conversion from bent to extended conformations.

We used negative stain electron microscopy (EM) to examine the conformational changes in the ectodomains required for activation of the leukocyte integrins alpha(X)beta(2) and alpha(L)beta(2). They transitioned between a bent conformation and two extended conformations in which the headpiece was in either a closed or an open state. Extended integrins exhibited marked flexibility at the alpha subunit genu and between integrin epidermal growth factor-like (I-EGF) domains 1 and 2. A clasp to mimic juxtamembrane association between the integrin alpha and beta subunits stabilized the bent conformation strongly for alpha(X)beta(2) and less so for alpha(L)beta(2). A small molecule allosteric antagonist induced the extended, open headpiece conformation. A Fab known to activate beta(2) integrins on leukocytes induced extension, and a Fab reporter of activation bound only after extension had been induced. The results establish an intimate relationship between extension of beta(2) integrins and their activation in immune responses and leukocyte trafficking.

Antibodies↗
PubMed2003

Collagen-binding mode of vWF-A3 domain determined by a transferred cross-saturation experiment.

The nature of the supramolecular complex between fibrillar collagen and collagen-binding proteins (CBPs) has hindered detailed X-ray and NMR analyses of the ligand-recognition mechanism at atomic resolution because of the lack of appropriate approaches for studying large heterogeneous supramolecular complexes. Recently, we proposed an NMR method, termed transferred cross-saturation (TCS), that enables the rigorous identification of contact residues in a huge protein complex. Here we used TCS to study the supramolecular complex between the A3 domain of von Willebrand factor and fibrillar collagen, which allowed the successful determination of the ligand-binding site of the A3 domain. The binding site of the A3 domain was located at its hydrophobic 'front' surface and was completely different from that of the I domain from the a2 subunit of integrin (alpha2-I domain), which was reported to be the hydrophilic 'top' surface of alpha2-I, although the A3 domain and the alpha2-I domain share a similar fold and possess the identical function of collagen binding.

Binding Sites↗