Limits...
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.

Show MeSH

Related in: MedlinePlus

Overlay of the 1H–15N HSQC spectra of L3-FKBP12 (a) and L4-FKBP12 (b) in the presence of one equivalent molar Lu3+ (blue), Dy3+ (green) and Tb3+ (black). Graphical views of the PCS isosurface of Dy3+ for L3-FKBP12 (c) and L4-FKBP12 (d). Positive and negative PCS values are indicated by blue and red, respectively. e Metal positions of L3- and L4-FKBP12. Metal positions are shown in ball (red for L3 and blue for L4)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351616&req=5

Fig2: Overlay of the 1H–15N HSQC spectra of L3-FKBP12 (a) and L4-FKBP12 (b) in the presence of one equivalent molar Lu3+ (blue), Dy3+ (green) and Tb3+ (black). Graphical views of the PCS isosurface of Dy3+ for L3-FKBP12 (c) and L4-FKBP12 (d). Positive and negative PCS values are indicated by blue and red, respectively. e Metal positions of L3- and L4-FKBP12. Metal positions are shown in ball (red for L3 and blue for L4)

Mentions: Considering the results of DSF and NMR analyses, we prepared three two-point anchored LBT-attached constructs, L3- to L5-FKBP12. Using these constructs, we examined the effect of spacer length on the principal axis of the Δχ-tensor and the metal position relative to the target protein. Figure 2a and b show the overlay spectra of Dy3+, Lu3+ and Tb3+-bound L3- (Fig. 2a) and L4-FKBP12-rapamycin (Fig. 2b), respectively. The peak shift pattern of L3-FKBP12-rapamycin was different from that of L4-FKBP12-rapamycin, showing differences in the Δχ-tensor. On the other hand, L4- and L5-FKBP12-rapamycin exhibited similar PCS values (Supplementary Fig. 2), and were assumed to exhibit similar Δχ-tensors and metal positions relative to the target protein. Hence, we estimated the Δχ-tensors for only L3- and L4-FKBP12-rapamycin from the PCS values using the Numbat program (Schmitz et al. 2008). For assignment of the PCS peaks, the 1H–15N HSQC spectrum of an eleven amino acid (A/F/H/I/K/L/M/R/V/W/Y) inversely labeled sample was also used to reduce spectral complexity (Supplementary Fig. 3). Based on the PCS values from the two lanthanide ions, Tb3+ and Dy3+, Δχ-tensors for each lanthanide were simultaneously fitted with the common metal position, due to their isomorphous nature, for L3- and L4-FKBP12-rapamycin, respectively (Table 2). The Δχ-tensors were well defined and the correlations between the experimental and back-calculated PCS values were good (Supplemental Fig. 4). This was also supported by the result of Monte-Carlo analysis using the 100 partial PCS data sets in which 30 % of the input data were randomly deleted (Supplemental Fig. 4). Moreover, the magnitudes of the tensors were comparable between L3- and L4-FKBP12-rapamycin as well as to those reported previously (for two-point anchored LBT-attached GB1; Saio et al. 2009, for the p62 PB1 domain; Saio et al. 2010 and for the Grb2 SH2 domain; Saio et al. 2011). Thus, we concluded that the positions of the lanthanide ions as well as the Δχ-tensor parameters for L3- and L4-FKBP12-rapamycin were accurately determined. In contrast to the similarity in magnitude of the Δχ-tensors between L3- and L4-FKBP12-rapamycin, the direction of the principal axes of the Δχ-tensors relative to the attached protein differed by about 30°–40° when compared with the same metal ion (Table 2; Fig. 2c, d). Moreover, the metal positions of these two constructs differed by about 5.2 Å (Fig. 2e). These observations suggest that the PCSs obtained from the two-point anchored LBT-attached proteins with different spacer lengths could be used as independent restraints for structural calculation.Fig. 2


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)

Overlay of the 1H–15N HSQC spectra of L3-FKBP12 (a) and L4-FKBP12 (b) in the presence of one equivalent molar Lu3+ (blue), Dy3+ (green) and Tb3+ (black). Graphical views of the PCS isosurface of Dy3+ for L3-FKBP12 (c) and L4-FKBP12 (d). Positive and negative PCS values are indicated by blue and red, respectively. e Metal positions of L3- and L4-FKBP12. Metal positions are shown in ball (red for L3 and blue for L4)
© Copyright Policy
Related In: Results  -  Collection

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

Fig2: Overlay of the 1H–15N HSQC spectra of L3-FKBP12 (a) and L4-FKBP12 (b) in the presence of one equivalent molar Lu3+ (blue), Dy3+ (green) and Tb3+ (black). Graphical views of the PCS isosurface of Dy3+ for L3-FKBP12 (c) and L4-FKBP12 (d). Positive and negative PCS values are indicated by blue and red, respectively. e Metal positions of L3- and L4-FKBP12. Metal positions are shown in ball (red for L3 and blue for L4)
Mentions: Considering the results of DSF and NMR analyses, we prepared three two-point anchored LBT-attached constructs, L3- to L5-FKBP12. Using these constructs, we examined the effect of spacer length on the principal axis of the Δχ-tensor and the metal position relative to the target protein. Figure 2a and b show the overlay spectra of Dy3+, Lu3+ and Tb3+-bound L3- (Fig. 2a) and L4-FKBP12-rapamycin (Fig. 2b), respectively. The peak shift pattern of L3-FKBP12-rapamycin was different from that of L4-FKBP12-rapamycin, showing differences in the Δχ-tensor. On the other hand, L4- and L5-FKBP12-rapamycin exhibited similar PCS values (Supplementary Fig. 2), and were assumed to exhibit similar Δχ-tensors and metal positions relative to the target protein. Hence, we estimated the Δχ-tensors for only L3- and L4-FKBP12-rapamycin from the PCS values using the Numbat program (Schmitz et al. 2008). For assignment of the PCS peaks, the 1H–15N HSQC spectrum of an eleven amino acid (A/F/H/I/K/L/M/R/V/W/Y) inversely labeled sample was also used to reduce spectral complexity (Supplementary Fig. 3). Based on the PCS values from the two lanthanide ions, Tb3+ and Dy3+, Δχ-tensors for each lanthanide were simultaneously fitted with the common metal position, due to their isomorphous nature, for L3- and L4-FKBP12-rapamycin, respectively (Table 2). The Δχ-tensors were well defined and the correlations between the experimental and back-calculated PCS values were good (Supplemental Fig. 4). This was also supported by the result of Monte-Carlo analysis using the 100 partial PCS data sets in which 30 % of the input data were randomly deleted (Supplemental Fig. 4). Moreover, the magnitudes of the tensors were comparable between L3- and L4-FKBP12-rapamycin as well as to those reported previously (for two-point anchored LBT-attached GB1; Saio et al. 2009, for the p62 PB1 domain; Saio et al. 2010 and for the Grb2 SH2 domain; Saio et al. 2011). Thus, we concluded that the positions of the lanthanide ions as well as the Δχ-tensor parameters for L3- and L4-FKBP12-rapamycin were accurately determined. In contrast to the similarity in magnitude of the Δχ-tensors between L3- and L4-FKBP12-rapamycin, the direction of the principal axes of the Δχ-tensors relative to the attached protein differed by about 30°–40° when compared with the same metal ion (Table 2; Fig. 2c, d). Moreover, the metal positions of these two constructs differed by about 5.2 Å (Fig. 2e). These observations suggest that the PCSs obtained from the two-point anchored LBT-attached proteins with different spacer lengths could be used as independent restraints for structural calculation.Fig. 2

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