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Grb7-SH2 domain dimerisation is affected by a single point mutation

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Abstract

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 μM. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.

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Abbreviations

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

EGF-R:

Epidermal growth factor receptor

Grb:

Growth factor bound protein

GST:

Glutathione S-transferase

Hepes:

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

IPTG:

Isopropyl-β-D-thiogalactopyranoside

MES:

2-Morpholinoethanesulfonic acid

PBS:

Phosphate-buffered saline

PH:

Plekstrin homology

PMSF:

Phenylmethylsulfonyl fluoride

SDS-PAGE:

Sodium dodecyl sulfate–polyacryamide gel electrophoresis

SH2:

Src homology 2

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Acknowledgements

We thank Krystal Ho and Loren Dyer for their contribution to this work. This research was supported by an Australian Research Council Fellowship and a Small University of Western Australia Research Grant awarded to J.A.W., as well as a UWA Hackett postgraduate scholarship awarded to C.J.P. J.P.M. is an NHMRC Senior Research Fellow.

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Authors and Affiliations

  1. School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, Australia

    Corrine J. Porter, Matthew C. J. Wilce & Jackie A. Wilce

  2. School of Medicine and Pharmacology, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, Australia

    Matthew C. J. Wilce & Peter Leedman

  3. Western Australian Institute for Medical Research, University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA, 6009, Australia

    Matthew C. J. Wilce & Peter Leedman

  4. School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW, 2006, Australia

    Joel P. Mackay

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  1. Corrine J. Porter
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Correspondence to Jackie A. Wilce.

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Porter, C.J., Wilce, M.C.J., Mackay, J.P. et al. Grb7-SH2 domain dimerisation is affected by a single point mutation. Eur Biophys J 34, 454–460 (2005). https://doi.org/10.1007/s00249-005-0480-1

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  • DOI: https://doi.org/10.1007/s00249-005-0480-1

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