NMR Study of the Structures of Repeated Sequences, GAGXGA (X = S, Y, V), in <i>Bombyx mori</i> Liquid Silk

The silk fibroin stored in the silk gland of the <i>Bombyx mori</i> silkworm, called “liquid silk”, is spun out and converted into the silk fiber with extremely high strength and high toughness. Therefore it is important to determine the silk structure before spinning called Silk I at an atomic level to clarify the fiber formation mechanism. We proposed the repeated type II β-turn structure as Silk I in the solid state with the model peptide (AG)₁₅ and several solid state NMR techniques previously. In this paper, the solution structure of native “liquid silk” was determined with solution NMR, especially for tandem repeated sequences with (GAGXGA)_<i>n</i> (X = S, Y, V) and GAASGA motifs in the <i>B. mori</i> silk fibroin. The assignment of the ¹³C, ¹⁵N, and ¹H solution NMR spectra for the repetitive sequence motifs was achieved, and the chemical shifts were obtained. The program, TALOS-N, to predict the backbone torsion angles from the chemical shifts of proteins was applied to these motifs with ¹³Cα, ¹³Cβ, ¹³CO, ¹Hα, ¹HN, and ¹⁵N chemical shifts. The twenty-five best matches of torsion angles (ϕ, φ) were well populated and mainly fell into the regions for typical type II β-turn structures in the (ϕ, φ) map for the GAGXGA (X = S, Y, V) motifs. In contrast, (ϕ, φ) plots for motif GAASGA were scattered, indicating that the motif is in a disordered structure. Furthermore, inter-residue HN-Hα NOE cross peaks between <i>i</i>-th and (<i>i</i>+2)­th residues in GAGXGA (X = S, Y, V) motifs were observed, supporting the repeated type II β-turn structure. Thus, we could show the presence of the repeated type II β-turn structure in “liquid silk”

Molecular basis for MPD binding to the JH-binding pocket of JHBP.

<p>(A) Interactions between JHBP and MPD1 bound in the JH-binding pocket observed for MPD-complex C. Residues involved in the recognition are shown as stick models and MPD1 as a ball-and-stick model. Hydrogen bond and CH-π stacking interactions used for recognition of MPD1 are indicated by light blue and orange dotted lines, respectively. (B) For comparison with (A), interactions of JHBP with the epoxy moiety of JH II observed for JH-complex A are displayed where JH II is shown as a ball-and-stick model. (C) Overlay of the JHBP-bound MPD1 (orange) and JH II (light blue) molecules. The red ball represents the oxygen atom which forms an intermolecular hydrogen bond with Tyr128 O^ηH of JHBP. (D) Chemical structures of JH II, MPD, methoprene, and methyl farnesoate (MF), the unepoxidated form of JH III, are shown from the top. The asterisk denotes a chiral carbon atom. (E) Competitive binding assay for MPD, methoprene and MF. The inhibition of JH binding to JHBP by the tested ligand was followed by monitoring the reduction of the radioactivity of the JHBP-bound ³H-labeled JH III. The relative radioactivity is plotted as a function of the ligand concentration: MPD (filled circles in red), methoprene (filled diamonds in green), and MF (filled squares in light-blue). The values represent the average for two to six experiments.</p

Summary of data collection and refinement statistics of the crystal structures of the JHBP-MPD complex.

a<p><i>R</i>_free-factors were calculated using 5% of the unique reflections.</p

MPD-induced conformational change in the second ligand-binding cavity of JHBP.

<p>(A) A close-up view of the MPD-bound second cavity in the JHBP-MPD complex. The figure is drawn for MPD-complex A, one of the four complexes in the crystal asymmetric unit. Residues involved in the recognition of MPD are shown as stick models and the bound ligand (MPD2) as a ball-and-stick model. A water molecule and hydrogen bonds are indicated by red sphere and light-blue dotted lines, respectively. (B) A close-up view of the unliganded second cavity in the JHBP-JH II complex shows that the side chain of Leu188 shown as a ball-and-stick model occupies the same space as MPD2 in the MPD-bound structure, caused by a kinked conformation of the α3 helix. The figure is drawn for JH-complex A, one of the two complexes in the crystal asymmetric unit. (C and D) The shapes of the MPD-bound second cavity and the unliganded second cavity calculated by the program GHECOM are shown as blue transparent shells, respectively. Original grid data of GHECOM represented by spheres are shown with white molecular surfaces of the proteins in insets.</p

Crystal structures of <i>Bombyx mori</i> JHBP.

<p>(A) A gate-open conformation of JHBP in the apo-form. The gate helix α1 shown in blue resides in an open conformation which permits access of JH to the preserved hormone-binding pocket. (B) A fully gate-closed conformation of JHBP in complex with JH II. The bound JH II molecule is shown as a space-filling model. The gate helix α1 shown in blue covers the hormone-binding pocket to maintain the bound JH II molecule deep inside the protein.</p

Comparison of the MPD-bound JHBP structure with the apo- and JH II-bound JHBP structures.

<p>(A) A side-view of the overlay of the crystal structures of the MPD-bound (yellow), JH II-bound (blue) and apo-JHBP (green), showing overall superposition. Significant deviations are observed at both ends of the elongated structure. The bound MPD and JH II molecules are shown as ball-and-stick models. (B) A top-view of the same overlay illustrates the difference around the JH-binding pocket. Axes of α1 helices are also shown. The orientation of the α1 helix on the JH-binding pocket, that functions as a gate sensing the ligand binding, is significantly different between the three states.</p

Structural plasticity of the JH-binding pocket in JHBP.

<p>(A) Views of the JH-binding pocket of the crystal structure of JHBP in complex with JH II. The figure is drawn for the JH-complex A, one of the two complexes in the crystal asymmetric unit. Surface representations of the complex structure were split vertically through the JH-binding pocket perpendicular to the page. The halves produced from the split were rotated in opposite directions to create the views shown. The interior of the protein and the exterior of the pocket are colored green and orange, respectively. The bound JH II molecule is shown as a ball-and-stick model with its molecular surface (pink). (B) Interactions between the latch-forming N-terminal arm and the C-terminal tail in the JH-bound JHBP observed for JH-complex A. The N-terminal arm and C-terminal tail are further linked to the gate α1 helix by the Cys9-Cys16 disulfide bond and the protein core by hydrogen bonds (light-blue dotted lines), respectively. Key residues for interactions are shown as stick models with hydrogen bonds (light-blue dotted lines). Leu6 in the N-terminal arm and Ser219 in the C-terminal tail are shown as space-filling models. The pocket is shown as a transparent orange surface with the internal JH II molecule as a ball-and-stick model. (C) Views of the JH-binding pocket of the crystal structure of JHBP in complex with MPD. The figure is drawn for MPD-complex C, one of the four complexes in the crystal asymmetric unit. Surface representations were created as for (A). The bound MPD molecule is shown as a ball-and-stick model with its molecular surface (pink). (D) Interactions between the latch-forming N-terminal arm and the C-terminal tail in the MPD-bound JHBP observed for MPD-complex C. As in the JH-complex, the N-terminal arm and C-terminal tail are further linked to the gate α1 helix by the Cys9-Cys16 disulfide bond and the protein core by hydrogen bonds (light-blue dotted lines), respectively. Key residues for interactions are shown as stick models with hydrogen bonds (light-blue dotted lines). Leu6 in the N-terminal arm and Ser219 in the C-terminal tail are shown as space-filling models. The pockets are shown as transparent orange surfaces with the internal MPD molecule as a ball-and-stick model.</p

Additional file 1: of A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

Primer sets used for PCR. (PDF 92 kb

Additional file 2: of A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity

Genotyping of FeDFR1a and FeDFR2. (A) Genotyping of FeDFR1a using PCR-amplified genomic DNA. A1 and A2 indicate the two alleles, and arrowheads indicate their positions. (B) Genotyping of FeDFR2 using the HincII digest of PCR-amplified genomic DNA. B1 and B2 indicate the two alleles, and arrowheads indicate their positions. (C) Sequence analysis of the FeDFR2 genomic DNA. An A to G transition at nucleotide 370, which results in a HincII restriction site in the FeDFR2 genome, is boxed in red. (D) Partial sequence alignment of FeDFR1aA1 and FeDFR1aA2. Identical nucleotides are highlighted in black. (PDF 68 kb