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Edited by Fred Dyda, National Institutes of Health, and accepted by Editorial Board Member Kiyoshi Mizuuchi September 29, 2017 (received for review August 16, 2017)
Article Figures & SI
Figures
- Fig. 1.
Overview of pNth11–751:Bmh1 structure. (A) Domain structure of yeast Nth1. The N-terminal extension and the calcium-binding domain (Nth1-CaBD) are shown in cyan, the catalytic domain (Nth1-CD) in dark cyan. The positions of PKA phosphorylation sites which are also 14-3-3 binding motifs are indicated with red arrows. The position of the EF-hand-like motif is indicated with its sequence. Residues involved in calcium ion coordination are shown in orange. Numbering is according to Nth1 from S. cerevisiae. (B) Structure of the pNth11–751:Bmh1 complex. The protomers of the Bmh1 homodimer are shown in yellow and brown. The N-terminal extension and the calcium-binding domain (Nth1-CaBD) are shown in cyan, the catalytic domain (Nth1-CD) in dark cyan. The phosphorylated S60 and S83 are shown in red. The calcium ion is shown in orange. (C) Close-up view of calcium ion-binding site.
- Fig. 2.
Contacts between Bmh1 and the 14-3-3 binding motifs of pNth11–751. (A) Contacts between Bmh1 and the first 14-3-3 binding motif of pNth11–751 containing pS60. The Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black. (B) Contacts between Bmh1 and the second 14-3-3 binding motif of pNth11–751 containing pS83. The Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black. (C) Contacts between Bmh1 and the intervening linker sequence between the two 14-3-3 binding motifs of pNth11–751. The omit 2F0 − Fc electron density map is contoured at 1σ. The Nth1 residues are labeled in cyan, the Bmh1 residues are labeled in black.
- Fig. 3.
Comparison of the Bmh1:pNth11–751, 14-3-3ζ:phosphopeptide, 14-3-3ζ:AANAT, and 14-3-3σ:heat shock protein beta-6 complexes. (A) Comparison of the main-chain conformation of the first 14-3-3 binding motif of pNth1 (sequence RTRTMpS60VFDN, shown in cyan) with a “mode 2” 14-3-3 peptide (sequence RLYHpSLPA, PDB ID 1QJA, shown in green) (25) and the 14-3-3 binding motifs of AANAT (sequence QRRHpTLPA, PDB ID 1IB1, shown in orange) (9) and heat shock protein beta-6 (sequence LRRApSAPL, PDB ID 5LTW, shown in red) (27). The C-terminal portion of the 14-3-3 binding motifs is indicated by black ellipse. (B) Comparison of the main-chain conformation of the second 14-3-3 binding motif of pNth1 (sequence QTRRGpS83EDDT, shown in cyan) with the same complexes as in A. (C and D) Comparison of the first 14-3-3 binding motif of pNth1 (sequence QTRRGpS83EDDT, shown in cyan) with a “mode 2” 14-3-3 peptide [sequence RLYHpSLPA, PDB ID 1QJA (25), shown in green] and the 14-3-3 binding motif of AANAT [sequence QRRHpTLPA, PDB ID 1IB1 (9), shown in orange]. The numbers describe the position relative to pS/pT.
- Fig. 4.
Other Bmh1:pNth11–751 interactions. (A) Close-up view of interactions between Nth1-CaBD (light cyan) and Bmh1 (yellow and brown). (B) Close-up view of interactions between the C-terminal helices A8 and A9 of Bmh1 (brown) with Nth1-CaBD (light cyan) and Nth1-CD (dark cyan).
- Fig. 5.
Structure of catalytic domain of Nth1. (A) Structure of catalytic domain of Bmh1-bound pNth11–751. The (α/α)6 barrel, subdomains I and II, and the C-terminal helices are shown in yellow, cyan, green, and blue, respectively. SUC is shown in gray. The C-terminal portion of the “lid” loop is shown in red. The rest of the structure is not shown. (B) Superimposition of the catalytic domain of Bmh1-bound pNth11–751 (blue), Nth1153–751 (green), and Nth1153–751:TRE (orange). The C-terminal portion of the “lid” loop of pNth11–751 is shown in red. (C) Close-up view of interactions between the “lid” loop and the EF-hand-like motif of Nth1-CaBD. (D) Sequence alignment of “lid” loops of yeast (S. cerevisiae) Nth1 and trehalase Tre37A from E. coli. The C-terminal portions of the “lid” loops are shown in red. (E) Close-up view of Nth1153–751 active site with bound trehalose (TRE). (F) Close-up view of active site of trehalase Tre37A from E. coli with bound inhibitor VDM (30).
- Fig. 6.
Time-resolved fluorescence experiments with DANS-TRE. (A) DANS-TRE. (B) Four tryptophan residues (W309, W624, W630, and W708) are located in close proximity to the substrate binding site of yeast Nth1. The molecule of trehalose (TRE) was modeled by superimposing the structure of Nth1153–751:TRE with Nth1-CD of Bmh1-bound pNth11–751. (C and D) Statistical confidence-interval analysis of intensity fraction of long fluorescence decay component corresponding to bound DANS-TRE in the presence of pNth11–751(E674A) (white), Bmh1 (light gray), and the pNth11–751(E674A):Bmh1complex (dark gray), with DANS-TRE excited at 290 (C) and 355 nm (D). Histograms represent the probability of recovering a particular intensity fraction from measured data as a result of 2,000 bootstrap fitting cycles (52). Dashed lines border 68% confidence intervals (1 SD). The histograms reveal that the intensity fraction of bound DANS-TRE is significantly higher in the presence of the pNth11–751(E674A):Bmh1complex compared with the control samples. The significantly increased bound-intensity fraction on excitation at 290 nm compared with excitation at 355 nm is likely a result of a combined effect of DANS-TRE FRET facilitated by Trp–DANS-TRE proximity in the binding pocket of the pNth11–751(E674A):Bmh1complex and blue shift of the absorption spectrum of bound DANS-TRE.
Data supplements
Supporting Information
Crystal structure of the 14-3-3:pNth1 complex
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