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

a Schematic representation of the two-point anchored LBT-attached FKBP12 construct. The spacer sequence is enclosed in the box. b The thermal unfolding curve of two-point anchored LBT-attached FKBP12 in the presence of one equivalent molar Lu3+. c Overlay of the 1H–15N HSQC spectra of FKBP12 (T75C) without the two-point anchored LBT (blue), and L1- (green), L2- (black), L3- (red), L4- (dark yellow) and L5-FKBP12 (gray) in the presence of 1 equivalent molar Lu3+. Inset shows peaks arising from G62. d Anchoring point, V2 and T75C (colored blue), and G62 (colored red) were mapped on the structure of FKBP12
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351616&req=5

Fig1: a Schematic representation of the two-point anchored LBT-attached FKBP12 construct. The spacer sequence is enclosed in the box. b The thermal unfolding curve of two-point anchored LBT-attached FKBP12 in the presence of one equivalent molar Lu3+. c Overlay of the 1H–15N HSQC spectra of FKBP12 (T75C) without the two-point anchored LBT (blue), and L1- (green), L2- (black), L3- (red), L4- (dark yellow) and L5-FKBP12 (gray) in the presence of 1 equivalent molar Lu3+. Inset shows peaks arising from G62. d Anchoring point, V2 and T75C (colored blue), and G62 (colored red) were mapped on the structure of FKBP12

Mentions: In the crystal structure of FKBP12 (Van Duyne et al. 1991), the well-defined secondary structure starts from V2. We, therefore, omitted G1 and defined the structured region of FKBP12 in all the two-point anchored LBT-attached FKBP12 constructs in this study. The distance between the Cα atoms of the N- and C-terminal residues is around 7 Å in the crystal structure of LBT (Nitz et al. 2004). We searched for a residue about 7 Å in distance from V2 of FKBP12, and found T75. The Cα distance between V2 and T75 was 5.6 Å. Thus we introduced the T75C mutation to FKBP12, and LBT was fused to the N-terminus of the FKBP12 (T75C). A spacer was introduced between the LBT and V2 of FKBP12 to avoid structural distortion and steric hindrance. We prepared constructs containing one- (H-), two- (H-M), three- (H-M-G), four- (H-M-S-G) and five-residue (H-M-G-S-G) linkers, named L1-, L2-, L3-, L4- and L5-FKBP12, respectively (Fig. 1a). These constructs were first screened for their suitability for NMR experiments, based on melting temperature (Tm) measured using differential scanning fluorometry (DSF; Niesen et al. 2007) in the presence of Lu3+, since we assumed that Tm was sensitive to the structural distortion and/or hindrance. Figure 1b shows the unfolding curves, and Table 1 lists the Tm values of Lu3+-bound L1- to L5-FKBP12-rapamycin. The unfolding curves of L3- to L5-FKBP12-rapamycin were almost identical, while those of L1- and L2-FKBP12-rapamycin were shifted to a lower temperature. The melting temperatures of L3- to L5-FKBP12-rapamycin were estimated to be around 72 °C, while those of L1- and L2-FKBP12-rapamycin were lower by 4 and 3.5 °C, respectively. From this observation, L1- and L2-FKBP12-rapamycin were assumed to exhibit structural distortion and/or hindrance. This was also confirmed by the comparison of the 1H–15N HSQC spectra of these constructs complexed with Lu3+ (Fig. 1c). The residues indicating spectral shifts on the attachment of the two-point anchored LBT were located very close to the anchoring points in the case of L3- to L5-FKBP12-rapamycin, while a large shift was observed for G62 (highlighted in Fig. 1d) on the α-helix region close to the N-terminal anchoring point in the case of L1- and L2-FKBP12-rapamycin. From NMR and DSF analyses, a linker with more than three amino acid residues was required for FKBP12 to avoid structural distortion and/or hindrance on the attachment of two-point anchored LBT.Fig. 1


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)

a Schematic representation of the two-point anchored LBT-attached FKBP12 construct. The spacer sequence is enclosed in the box. b The thermal unfolding curve of two-point anchored LBT-attached FKBP12 in the presence of one equivalent molar Lu3+. c Overlay of the 1H–15N HSQC spectra of FKBP12 (T75C) without the two-point anchored LBT (blue), and L1- (green), L2- (black), L3- (red), L4- (dark yellow) and L5-FKBP12 (gray) in the presence of 1 equivalent molar Lu3+. Inset shows peaks arising from G62. d Anchoring point, V2 and T75C (colored blue), and G62 (colored red) were mapped on the structure of FKBP12
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: a Schematic representation of the two-point anchored LBT-attached FKBP12 construct. The spacer sequence is enclosed in the box. b The thermal unfolding curve of two-point anchored LBT-attached FKBP12 in the presence of one equivalent molar Lu3+. c Overlay of the 1H–15N HSQC spectra of FKBP12 (T75C) without the two-point anchored LBT (blue), and L1- (green), L2- (black), L3- (red), L4- (dark yellow) and L5-FKBP12 (gray) in the presence of 1 equivalent molar Lu3+. Inset shows peaks arising from G62. d Anchoring point, V2 and T75C (colored blue), and G62 (colored red) were mapped on the structure of FKBP12
Mentions: In the crystal structure of FKBP12 (Van Duyne et al. 1991), the well-defined secondary structure starts from V2. We, therefore, omitted G1 and defined the structured region of FKBP12 in all the two-point anchored LBT-attached FKBP12 constructs in this study. The distance between the Cα atoms of the N- and C-terminal residues is around 7 Å in the crystal structure of LBT (Nitz et al. 2004). We searched for a residue about 7 Å in distance from V2 of FKBP12, and found T75. The Cα distance between V2 and T75 was 5.6 Å. Thus we introduced the T75C mutation to FKBP12, and LBT was fused to the N-terminus of the FKBP12 (T75C). A spacer was introduced between the LBT and V2 of FKBP12 to avoid structural distortion and steric hindrance. We prepared constructs containing one- (H-), two- (H-M), three- (H-M-G), four- (H-M-S-G) and five-residue (H-M-G-S-G) linkers, named L1-, L2-, L3-, L4- and L5-FKBP12, respectively (Fig. 1a). These constructs were first screened for their suitability for NMR experiments, based on melting temperature (Tm) measured using differential scanning fluorometry (DSF; Niesen et al. 2007) in the presence of Lu3+, since we assumed that Tm was sensitive to the structural distortion and/or hindrance. Figure 1b shows the unfolding curves, and Table 1 lists the Tm values of Lu3+-bound L1- to L5-FKBP12-rapamycin. The unfolding curves of L3- to L5-FKBP12-rapamycin were almost identical, while those of L1- and L2-FKBP12-rapamycin were shifted to a lower temperature. The melting temperatures of L3- to L5-FKBP12-rapamycin were estimated to be around 72 °C, while those of L1- and L2-FKBP12-rapamycin were lower by 4 and 3.5 °C, respectively. From this observation, L1- and L2-FKBP12-rapamycin were assumed to exhibit structural distortion and/or hindrance. This was also confirmed by the comparison of the 1H–15N HSQC spectra of these constructs complexed with Lu3+ (Fig. 1c). The residues indicating spectral shifts on the attachment of the two-point anchored LBT were located very close to the anchoring points in the case of L3- to L5-FKBP12-rapamycin, while a large shift was observed for G62 (highlighted in Fig. 1d) on the α-helix region close to the N-terminal anchoring point in the case of L1- and L2-FKBP12-rapamycin. From NMR and DSF analyses, a linker with more than three amino acid residues was required for FKBP12 to avoid structural distortion and/or hindrance on the attachment of two-point anchored LBT.Fig. 1

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