The high-resolution NMR structure of the R21A Spc-SH3:P41 complex

Understanding the determinants of binding affinity by comparison with Abl-SH3

Salvador Casares, Eiso Ab, Hendrik Eshuis, Obdulio Lopez-Mayorga, Nico A.J. Van Nuland, Francisco Conejero-Lara

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Abstract

Background. SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3). Results. Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. Conclusion. Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.

Original languageEnglish
Article number22
JournalBMC Structural Biology
Volume7
DOIs
StatePublished - 27 Apr 2007

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Spectrin
src Homology Domains
Thermodynamics
Ligands
Arginine
Proteins
Water
Deuterium
Drug Design
Differential Scanning Calorimetry
Entropy
Proline
Amides
Alanine
Protein-Tyrosine Kinases
Hydrogen
Acquired Immunodeficiency Syndrome
Binding Sites

Cite this

Casares, Salvador ; Ab, Eiso ; Eshuis, Hendrik ; Lopez-Mayorga, Obdulio ; Van Nuland, Nico A.J. ; Conejero-Lara, Francisco. / The high-resolution NMR structure of the R21A Spc-SH3:P41 complex : Understanding the determinants of binding affinity by comparison with Abl-SH3. In: BMC Structural Biology. 2007 ; Vol. 7.
@article{7c29072f44364e54a02295e15b7446ab,
title = "The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3",
abstract = "Background. SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3). Results. Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. Conclusion. Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.",
author = "Salvador Casares and Eiso Ab and Hendrik Eshuis and Obdulio Lopez-Mayorga and {Van Nuland}, {Nico A.J.} and Francisco Conejero-Lara",
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The high-resolution NMR structure of the R21A Spc-SH3:P41 complex : Understanding the determinants of binding affinity by comparison with Abl-SH3. / Casares, Salvador; Ab, Eiso; Eshuis, Hendrik; Lopez-Mayorga, Obdulio; Van Nuland, Nico A.J.; Conejero-Lara, Francisco.

In: BMC Structural Biology, Vol. 7, 22, 27.04.2007.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The high-resolution NMR structure of the R21A Spc-SH3:P41 complex

T2 - Understanding the determinants of binding affinity by comparison with Abl-SH3

AU - Casares, Salvador

AU - Ab, Eiso

AU - Eshuis, Hendrik

AU - Lopez-Mayorga, Obdulio

AU - Van Nuland, Nico A.J.

AU - Conejero-Lara, Francisco

PY - 2007/4/27

Y1 - 2007/4/27

N2 - Background. SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3). Results. Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. Conclusion. Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.

AB - Background. SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3). Results. Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. Conclusion. Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.

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U2 - 10.1186/1472-6807-7-22

DO - 10.1186/1472-6807-7-22

M3 - Article

VL - 7

JO - BMC Structural Biology

JF - BMC Structural Biology

SN - 1472-6807

M1 - 22

ER -