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Article

Structural and Thermodynamic Characterization of the Interaction of the SH3 Domain from Fyn with the Proline-Rich Binding Site on the p85 Subunit of PI3-Kinase^†

D. A. Renzoni,^‡^§ D. J. R. Pugh,^§ G. Siligardi, P. Das,^# C. J. Morton, C. Rossi,^‡ M. D. Waterfield,^# I. D. Campbell, and J. E. Ladbury*

Oxford Centre for Molecular Science, University of Oxford, South Parks Road, Oxford OX1 3QT, U.K., Dipartimento di Chimica, Universit di Siena, Pian dei Mantellini 44, 53100 Siena, Italy, National Chiroptical and Optical Spectroscopy Facility, Department of Pharmacy, King's College London, Manresa Road, London SW3 6LX, U.K., and Ludwig Institute for Cancer Research and Department of Biochemistry and Molecular Biology, University College London, 91 Riding House Street, London W1P 8BT, U.K.

Biochemistry, 1996, 35 (49), pp 15646–15653

DOI: 10.1021/bi9620969

Publication Date (Web): December 10, 1996

†

D.J.R.P. is a Wellcome Trust Mathematical Biology Fellow and J.E.L. is Wellcome Trust Research Career Development Fellow. We thank the Wellcome Trust and Zeneca for support for I.D.C. and C.J.M., respectively. The OCMS is funded by the BBSRC, MRC, and EPSRC. The NCOSC is funded by the EPSRC and ULIRS.

‡

Università di Siena.

Authors contributed equally to this work.

University of Oxford.

King's College London.

University College London.

Address correspondence to this author.

Present address: Department of Biochemistry and Molecular Biology, University College London, 91 Riding House St., London W1P 8BT, U.K.

Abstract

The interaction of the Fyn SH3 domain with the p85 subunit of PI3-kinase is investigated using structural detail and thermodynamic data. The solution structure complex of the SH3 domain with a proline-rich peptide mimic of the binding site on the p85 subunit is described. This indicates that the peptide binds as a poly(l-proline) type II helix. Circular dichroism spectroscopic studies reveal that in the unbound state the peptide exhibits no structure. Thermodynamic data for the binding of this peptide to the SH3 domain suggest that the weak binding (approximately 31 μM) of this interaction is, in part, due to the entropically unfavorable effect of helix formation (ΔS° = −78 J·mol^-¹·K^-¹). Binding of the SH3 domain to the intact p85 subunit (minus its own SH3 domain) is tighter, and the entropic and enthalpic contributions are very different from those given by the peptide interaction (ΔS° = +252 J·mol^-¹·K^-¹; ΔH° = +44 kJ·mol^-¹). From these dramatically different thermodynamic measurements we are able to conclude that the interaction of the proline-rich peptide does not effectively mimic the interaction of the intact p85 subunit with the SH3 domain and suggest that other interactions could be important.