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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 P3Tm and SH3 (shown in green). (b) Violations analysis of calculated position of TOAC in P3Tm 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 class 2 residues: distance obtained in rigid-body docking calculations versus PRE-derived distance. (d) Same as (a), overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). In purple is shown the residue in peptide RALPPLPRY that corresponds most closely to the position of TOAC in peptide P3Tm
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Fig6: (a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Tm and SH3 (shown in green). (b) Violations analysis of calculated position of TOAC in P3Tm 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 class 2 residues: distance obtained in rigid-body docking calculations versus PRE-derived distance. (d) Same as (a), overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). In purple is shown the residue in peptide RALPPLPRY that corresponds most closely to the position of TOAC in peptide P3Tm

Mentions: After deprotonation of the TOAC nitroxide, peptides were added to 15N-labelled Src SH3 domain, causing a decrease in intensity for some residues (Fig. 5). Distance restraints were calculated from the NMR data and used in docking calculations (supplementary material). For peptide P3Tm multiple rigid-body docking runs with random starting positions for the TOAC nitroxide oxygen atom consistently produced a single low-energy solution (Fig. 6a). Analysis of the solution shows that virtually all restraints are satisfied and that the position of the spin-label is well-defined (Fig. 6b). Any violations observed can be explained by small movements of residues situated in more flexible regions of the protein. To measure the agreement between the observed and calculated distances a Q-factor was calculated for the double-bounded restraints (see Experimental Procedures). For peptide P3Tm the Q-factor was 0.08 with a correlation coefficient of 0.96 (Fig. 6c). The calculated position of the spin-label is reasonable as judged from comparison with a structure of chicken Src SH3 domain in complex with a similar peptide (Fig. 6d). It should be noted that the introduction of the TOAC can have distorted the peptide, given the large reduction in the affinity.Fig. 5


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 P3Tm and SH3 (shown in green). (b) Violations analysis of calculated position of TOAC in P3Tm 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 class 2 residues: distance obtained in rigid-body docking calculations versus PRE-derived distance. (d) Same as (a), overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). In purple is shown the residue in peptide RALPPLPRY that corresponds most closely to the position of TOAC in peptide P3Tm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480485&req=5

Fig6: (a) Calculated position of TOAC oxygen atom, shown as pink sphere, in complex between peptide P3Tm and SH3 (shown in green). (b) Violations analysis of calculated position of TOAC in P3Tm 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 class 2 residues: distance obtained in rigid-body docking calculations versus PRE-derived distance. (d) Same as (a), overlaid with structure of SH3 in complex with peptide RALPPLPRY, shown in yellow (PDB entry 1RLQ). In purple is shown the residue in peptide RALPPLPRY that corresponds most closely to the position of TOAC in peptide P3Tm
Mentions: After deprotonation of the TOAC nitroxide, peptides were added to 15N-labelled Src SH3 domain, causing a decrease in intensity for some residues (Fig. 5). Distance restraints were calculated from the NMR data and used in docking calculations (supplementary material). For peptide P3Tm multiple rigid-body docking runs with random starting positions for the TOAC nitroxide oxygen atom consistently produced a single low-energy solution (Fig. 6a). Analysis of the solution shows that virtually all restraints are satisfied and that the position of the spin-label is well-defined (Fig. 6b). Any violations observed can be explained by small movements of residues situated in more flexible regions of the protein. To measure the agreement between the observed and calculated distances a Q-factor was calculated for the double-bounded restraints (see Experimental Procedures). For peptide P3Tm the Q-factor was 0.08 with a correlation coefficient of 0.96 (Fig. 6c). The calculated position of the spin-label is reasonable as judged from comparison with a structure of chicken Src SH3 domain in complex with a similar peptide (Fig. 6d). It should be noted that the introduction of the TOAC can have distorted the peptide, given the large reduction in the affinity.Fig. 5

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