Protein purification and the oligomerization analysis of the NSF mutants.

<p>(A) Purity of the NSF proteins. The final purified proteins (3 μg of each proteins) were resolved by 13.5% SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. The molecular size standards are shown on the left. (B) Oligomeric state analysis by size-exclusion chromatography in the buffer containing ADP-AlFx. The typical profiles of NSF (WT), E203A (a representative mutant of N-terminal region), P211A (a representative mutant of middle region), G221PG222P (a representative mutant of C-terminal region), N210A and E216A (the mutants that show the strongest effect in SNARE disassembly assay) from a Superose-6 column were present.</p

Summary of SNARE disassembly and ATPase activities of NSF N-D1 linker mutants.

<p>SNARE disassembly, basal and stimulated ATPase activities of wild-type NSF were set as 1.00. The values for the mutants were normalized to wild-type NSF.</p

Mutational effects of the N-terminal region of the NSF N-D1 linker.

<p>(A) SNARE disassembly by wild-type and mutant NSF. SNARE complexes were incubated with wild-type or mutant NSF, and α-SNAP in the presence of Mg²⁺-ATP at 37°C for 0 min, 20 min and 60 min, followed by the addition of SDS-PAGE loading buffer and analyzed by SDS-PAGE. The SNARE proteins were quantified by densitometry using ImageJ. The histogram shows the SNARE disassembly activities of wild-type and mutated NSF at 60 min averaged from three independent measurements and calculated as follows: disassembled protein, obtained by subtracting remaining protein (60 min) from total protein (0 min), divided by total protein (0 min). Values have been normalized to that of wild-type (WT) NSF. Error bars indicate the standard deviation. (B) Binding of wild-type and mutant NSF to the SNARE/α-SNAP complex. Wild-type or mutant NSF proteins were incubated with MBP-SNARE complexes in the presence (+) or absence (−) of α-SNAP under the ADP-AlFx state. The bound proteins were collected with amylose magnetic beads, eluted with 10 mM maltose and analyzed by SDS-PAGE. The gels (left panel) presented are representative of at least two separate experiments. The bound proteins were quantified by densitometry using ImageJ (right panel). (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF. Standard ATPase reactions were carried out using 3 μg of wild-type or mutant NSF in the ATPase assay buffer. SNARE/α-SNAP complex was prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064346#s4" target="_blank">Materials and Methods</a>. Basal and stimulated ATPase activities were measured in the absence (−) or presence (+) of SNARE/α-SNAP complex at 25°C for 1 h, respectively. The histogram shows the rates of ATP hydrolysis averaged from three independent measurements. Error bars indicate standard deviations. The fold increase in ATPase activity (stimulation) was calculated by dividing the stimulated ATPase activity by the basal ATPase activity.</p

Domain sketch of NSF protein and the amino acid conservation of the N-D1 linker.

<p>The diagram shows the structural domains of NSF: the N-terminal domain, D1 and D2 AAA+ modules. The position of the N-D1 linker and D1-D2 linker are indicated. Sequence alignment of the N-D1 linker from different eukaryotic species shows that the N-D1 linker is highly conserved. The numbers at the two ends of the sequence indicate the amino acid residues in NSF. Residues are colored by similarity. The letters under the sequence indicate the amino acid substitutions constructed in this study.</p

Mutational effects of the middle region of the NSF N-D1 linker.

<p>(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.</p

Mutational effects of the C-terminal region of the NSF N-D1 linker.

<p>(A) SNARE disassembly by wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3A. (B) Binding of wild-type and mutant NSF proteins to the SNARE/α-SNAP complex. Measurements are the same as in Fig. 3B. (C) Basal and SNARE/α-SNAP stimulated ATPase activities of wild-type and mutant NSF proteins. Measurements and presentation of results are the same as in Fig. 3C.</p

Heterologous Expression and Characterization of a Novel Chitinase (ChiEn1) from <i>Coprinopsis cinerea</i> and its Synergism in the Degradation of Chitin

Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation activities toward chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the released reducing sugars the amount of (GlcNAc)₂ was increased by 149.5%, whereas the amount of GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1, rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action

The Modes of Action of ChiIII, a Chitinase from Mushroom Coprinopsis cinerea, Shift with Changes in the Length of GlcNAc Oligomers

A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)_3–5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)_3–5; when the substrate was (GlcNAc)_6–7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+β domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities