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Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR.

Lindfors HE, de Koning PE, Drijfhout JW, Venezia B, Ubbink M - J. Biomol. NMR (2008)

Bottom Line: Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially.Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined.It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

View Article: PubMed Central - PubMed

Affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.

ABSTRACT
Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

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(a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Te and SH3 (in green, residue D10 is shown in purple). Overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). The N-terminus of the peptide, coloured blue, corresponds to the place of attachment of the TOAC amino acid in peptide P3Te. (b) Violations analysis of calculated position of TOAC in P3Te in complex with SH3. Dotted line: PRE-derived distance; white circles: distance in calculated structure; shaded area: error margins used in calculations. For class 1 residues (upper bound only) the error margin was +2Å, for class 3 residues (lower bound only) a −2Å error margin was used, and for class 2 residues (both upper and lower distance restraints) the error margins were ± 1 Å. (c) Distance from TOAC oxygen atom to backbone amide proton for residues with both upper and lower distance restraints (class 2): distance obtained in rigid-body docking calculations versus PRE-derived distance
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Fig8: (a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Te and SH3 (in green, residue D10 is shown in purple). Overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). The N-terminus of the peptide, coloured blue, corresponds to the place of attachment of the TOAC amino acid in peptide P3Te. (b) Violations analysis of calculated position of TOAC in P3Te in complex with SH3. Dotted line: PRE-derived distance; white circles: distance in calculated structure; shaded area: error margins used in calculations. For class 1 residues (upper bound only) the error margin was +2Å, for class 3 residues (lower bound only) a −2Å error margin was used, and for class 2 residues (both upper and lower distance restraints) the error margins were ± 1 Å. (c) Distance from TOAC oxygen atom to backbone amide proton for residues with both upper and lower distance restraints (class 2): distance obtained in rigid-body docking calculations versus PRE-derived distance

Mentions: Rigid-body docking calculations for the position of TOAC in the complex of SH3 and peptide P3Te also yields a single, reproducible solution (Fig. 8a). Analysis of the solution, however, shows violations for several residues (Fig. 8b). A Q-factor of 0.17 was calculated together with a correlation coefficient of merely 0.62 (Fig. 8c). Closer inspection of the data shows that the poor fit is largely due to one residue, D10 (corresponding to residue D93 in full-length mouse Src). This residue has an intensity ratio of 0.70, but is located far from the rest of the residues that exhibit an effect from the spin-label, and pulls the calculated final position of the spin-label away from the other residues. Excluding this outlier from the docking calculations changes the final position of the TOAC oxygen atom, the TOAC is now positioned further away from the centre of the peptide in a more realistic position (Fig. 9a). This improves the fit, with a new Q-factor of 0.13 and a correlation coefficient of 0.81 (Fig. 9b,c). There are still regions where the observed PRE effect is slightly larger than expected from the calculated structure. This is typically seen in systems where dynamics is present (Volkov et al. 2006) and can be accounted for by small movements of the spin-label placed at one end of the peptide, where the flexibility is higher.Fig. 8


Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR.

Lindfors HE, de Koning PE, Drijfhout JW, Venezia B, Ubbink M - J. Biomol. NMR (2008)

(a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Te and SH3 (in green, residue D10 is shown in purple). Overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). The N-terminus of the peptide, coloured blue, corresponds to the place of attachment of the TOAC amino acid in peptide P3Te. (b) Violations analysis of calculated position of TOAC in P3Te in complex with SH3. Dotted line: PRE-derived distance; white circles: distance in calculated structure; shaded area: error margins used in calculations. For class 1 residues (upper bound only) the error margin was +2Å, for class 3 residues (lower bound only) a −2Å error margin was used, and for class 2 residues (both upper and lower distance restraints) the error margins were ± 1 Å. (c) Distance from TOAC oxygen atom to backbone amide proton for residues with both upper and lower distance restraints (class 2): distance obtained in rigid-body docking calculations versus PRE-derived distance
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Fig8: (a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Te and SH3 (in green, residue D10 is shown in purple). Overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). The N-terminus of the peptide, coloured blue, corresponds to the place of attachment of the TOAC amino acid in peptide P3Te. (b) Violations analysis of calculated position of TOAC in P3Te in complex with SH3. Dotted line: PRE-derived distance; white circles: distance in calculated structure; shaded area: error margins used in calculations. For class 1 residues (upper bound only) the error margin was +2Å, for class 3 residues (lower bound only) a −2Å error margin was used, and for class 2 residues (both upper and lower distance restraints) the error margins were ± 1 Å. (c) Distance from TOAC oxygen atom to backbone amide proton for residues with both upper and lower distance restraints (class 2): distance obtained in rigid-body docking calculations versus PRE-derived distance
Mentions: Rigid-body docking calculations for the position of TOAC in the complex of SH3 and peptide P3Te also yields a single, reproducible solution (Fig. 8a). Analysis of the solution, however, shows violations for several residues (Fig. 8b). A Q-factor of 0.17 was calculated together with a correlation coefficient of merely 0.62 (Fig. 8c). Closer inspection of the data shows that the poor fit is largely due to one residue, D10 (corresponding to residue D93 in full-length mouse Src). This residue has an intensity ratio of 0.70, but is located far from the rest of the residues that exhibit an effect from the spin-label, and pulls the calculated final position of the spin-label away from the other residues. Excluding this outlier from the docking calculations changes the final position of the TOAC oxygen atom, the TOAC is now positioned further away from the centre of the peptide in a more realistic position (Fig. 9a). This improves the fit, with a new Q-factor of 0.13 and a correlation coefficient of 0.81 (Fig. 9b,c). There are still regions where the observed PRE effect is slightly larger than expected from the calculated structure. This is typically seen in systems where dynamics is present (Volkov et al. 2006) and can be accounted for by small movements of the spin-label placed at one end of the peptide, where the flexibility is higher.Fig. 8

Bottom Line: Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially.Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined.It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

View Article: PubMed Central - PubMed

Affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands.

ABSTRACT
Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

Show MeSH
Related in: MedlinePlus