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Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination.

Kobashigawa Y, Saio T, Ushio M, Sekiguchi M, Yokochi M, Ogura K, Inagaki F - J. Biomol. NMR (2012)

Bottom Line: We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins.Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein.We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, N-21, W-11, Sapporo 001-0021, Japan.

ABSTRACT
Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

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The PCS-based docking structure between the FKBP12-rapamycin and FRB domains. Since PCS data were not obtained for rapamycin, rapamycin was omitted during the structure calculation. a Calculated FKBP12-FRB complex structure based on PCS data from L3-FKBP12 using two metals, both Dy3+ and Tb3+. b Calculated FKBP12-FRB complex structure based on PCS data from L4-FKBP12 using two metals, both Dy3+ and Tb3+. c Calculated FKBP12-FRB complex structure based on PCS data from both L3- and L4-FKBP12 using two metals, both Dy3+ and Tb3+. Through (a) to (c), obtained structures were superimposed on FKBP12 moiety. In (a), (b) and (c), metal positions are shown in ball (red for L3 and blue for L4), FKBP12 in green ribbon and FRB in magenta stick. dRibbon representation of the PCS-based structure (green for FKBP12 and magenta for FRB) and the crystal structure of FKBP12/rapamycin/FRB ternary complex (Liang et al. 1999, 1fap.pdb; cyan ribbon for FKBP12, orange stick for rapamycin and blue ribbon for FRB). The lowest energy structure of the PCS-based structure of the FKBP12-FRB complex was superimposed on FKBP12 moiety of the crystal structure of the ternary complex (Liang et al. 1999, 1fap.pdb). The main chain atom RMSD of the FKBP12 moiety in the binary and the ternary complexes was estimated to be 0.5 Å. The main chain atom RMSD of the FRB moiety in the ternary complex of the crystal structure and the PCS-based NMR structure was estimated to be 2.9 Å
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Fig3: The PCS-based docking structure between the FKBP12-rapamycin and FRB domains. Since PCS data were not obtained for rapamycin, rapamycin was omitted during the structure calculation. a Calculated FKBP12-FRB complex structure based on PCS data from L3-FKBP12 using two metals, both Dy3+ and Tb3+. b Calculated FKBP12-FRB complex structure based on PCS data from L4-FKBP12 using two metals, both Dy3+ and Tb3+. c Calculated FKBP12-FRB complex structure based on PCS data from both L3- and L4-FKBP12 using two metals, both Dy3+ and Tb3+. Through (a) to (c), obtained structures were superimposed on FKBP12 moiety. In (a), (b) and (c), metal positions are shown in ball (red for L3 and blue for L4), FKBP12 in green ribbon and FRB in magenta stick. dRibbon representation of the PCS-based structure (green for FKBP12 and magenta for FRB) and the crystal structure of FKBP12/rapamycin/FRB ternary complex (Liang et al. 1999, 1fap.pdb; cyan ribbon for FKBP12, orange stick for rapamycin and blue ribbon for FRB). The lowest energy structure of the PCS-based structure of the FKBP12-FRB complex was superimposed on FKBP12 moiety of the crystal structure of the ternary complex (Liang et al. 1999, 1fap.pdb). The main chain atom RMSD of the FKBP12 moiety in the binary and the ternary complexes was estimated to be 0.5 Å. The main chain atom RMSD of the FRB moiety in the ternary complex of the crystal structure and the PCS-based NMR structure was estimated to be 2.9 Å

Mentions: We next studied whether the present approach could be used for resolving the degeneracy problem in PCS-based structure calculation. We initially confirmed that FRB moiety of FRB/rapamycin/L3-FKBP12 ternary complex exhibited different PCS pattern as compared to the FRB/rapamycin/L4-FKBP12 (Supplementary Fig. 7). Next, the structure of the FKBP12-FRB complex was calculated based solely on PCS restraints, and compared with the crystal structure (Liang et al. 1999). First, rigid body docking calculations were performed for L3- and L4-FKBP12 separately, using two PCS data sets derived from Dy3+ and Tb3+. The docking structure determined using PCS data sets derived from two lanthanide ions still affords four degenerate solutions (Fig. 3a, b). This is consistent with our previous result (Saio et al. 2010).Fig. 3


Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination.

Kobashigawa Y, Saio T, Ushio M, Sekiguchi M, Yokochi M, Ogura K, Inagaki F - J. Biomol. NMR (2012)

The PCS-based docking structure between the FKBP12-rapamycin and FRB domains. Since PCS data were not obtained for rapamycin, rapamycin was omitted during the structure calculation. a Calculated FKBP12-FRB complex structure based on PCS data from L3-FKBP12 using two metals, both Dy3+ and Tb3+. b Calculated FKBP12-FRB complex structure based on PCS data from L4-FKBP12 using two metals, both Dy3+ and Tb3+. c Calculated FKBP12-FRB complex structure based on PCS data from both L3- and L4-FKBP12 using two metals, both Dy3+ and Tb3+. Through (a) to (c), obtained structures were superimposed on FKBP12 moiety. In (a), (b) and (c), metal positions are shown in ball (red for L3 and blue for L4), FKBP12 in green ribbon and FRB in magenta stick. dRibbon representation of the PCS-based structure (green for FKBP12 and magenta for FRB) and the crystal structure of FKBP12/rapamycin/FRB ternary complex (Liang et al. 1999, 1fap.pdb; cyan ribbon for FKBP12, orange stick for rapamycin and blue ribbon for FRB). The lowest energy structure of the PCS-based structure of the FKBP12-FRB complex was superimposed on FKBP12 moiety of the crystal structure of the ternary complex (Liang et al. 1999, 1fap.pdb). The main chain atom RMSD of the FKBP12 moiety in the binary and the ternary complexes was estimated to be 0.5 Å. The main chain atom RMSD of the FRB moiety in the ternary complex of the crystal structure and the PCS-based NMR structure was estimated to be 2.9 Å
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351616&req=5

Fig3: The PCS-based docking structure between the FKBP12-rapamycin and FRB domains. Since PCS data were not obtained for rapamycin, rapamycin was omitted during the structure calculation. a Calculated FKBP12-FRB complex structure based on PCS data from L3-FKBP12 using two metals, both Dy3+ and Tb3+. b Calculated FKBP12-FRB complex structure based on PCS data from L4-FKBP12 using two metals, both Dy3+ and Tb3+. c Calculated FKBP12-FRB complex structure based on PCS data from both L3- and L4-FKBP12 using two metals, both Dy3+ and Tb3+. Through (a) to (c), obtained structures were superimposed on FKBP12 moiety. In (a), (b) and (c), metal positions are shown in ball (red for L3 and blue for L4), FKBP12 in green ribbon and FRB in magenta stick. dRibbon representation of the PCS-based structure (green for FKBP12 and magenta for FRB) and the crystal structure of FKBP12/rapamycin/FRB ternary complex (Liang et al. 1999, 1fap.pdb; cyan ribbon for FKBP12, orange stick for rapamycin and blue ribbon for FRB). The lowest energy structure of the PCS-based structure of the FKBP12-FRB complex was superimposed on FKBP12 moiety of the crystal structure of the ternary complex (Liang et al. 1999, 1fap.pdb). The main chain atom RMSD of the FKBP12 moiety in the binary and the ternary complexes was estimated to be 0.5 Å. The main chain atom RMSD of the FRB moiety in the ternary complex of the crystal structure and the PCS-based NMR structure was estimated to be 2.9 Å
Mentions: We next studied whether the present approach could be used for resolving the degeneracy problem in PCS-based structure calculation. We initially confirmed that FRB moiety of FRB/rapamycin/L3-FKBP12 ternary complex exhibited different PCS pattern as compared to the FRB/rapamycin/L4-FKBP12 (Supplementary Fig. 7). Next, the structure of the FKBP12-FRB complex was calculated based solely on PCS restraints, and compared with the crystal structure (Liang et al. 1999). First, rigid body docking calculations were performed for L3- and L4-FKBP12 separately, using two PCS data sets derived from Dy3+ and Tb3+. The docking structure determined using PCS data sets derived from two lanthanide ions still affords four degenerate solutions (Fig. 3a, b). This is consistent with our previous result (Saio et al. 2010).Fig. 3

Bottom Line: We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins.Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein.We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, N-21, W-11, Sapporo 001-0021, Japan.

ABSTRACT
Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

Show MeSH
Related in: MedlinePlus