Mechanism of Oligomerisation of Cyclase-associated Protein from Dictyostelium discoideum in Solution

Mechanism of Oligomerisation of Cyclase-associated Protein from Dictyostelium discoideum in Solution
Received 27 June 2006; accepted 1 August 2006. Edited by R. Huber. Available online 4 August 2006.
Adlina Mohd Yusof1, Elmar Jaenicke2, Jan Skov Pedersen3, Angelika A. Noegel4, Michael Schleicher5 and Andreas Hofmann1, , 1
Journal of Molecular Biology
Volume 362, Issue 5 , 6 October 2006
Copyright ? 2006 Elsevier Ltd All rights reserved.
1Institute of Structural and Molecular Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, UK
2Institute for Molecular Biophysics, Johannes-Gutenberg Universit?t, 55099 Mainz, Germany
3Department of Chemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
4Center for Biochemistry, Medical Faculty, Universit?t zu K?ln, 50931 K?ln, Germany
5Institute of Cell Biology, Ludwig-Maximilians Universit?t, 80336 M?nchen, Germany
Cyclase-associated protein (CAP) is a highly conserved modular protein implicated in the regulation of actin filament dynamics and a variety of developmental and morphological processes. The protein exists as a high molecular weight complex in cell extracts and purified protein possesses a high tendency to aggregate, a major obstacle for crystallisation. Using a mutagenesis approach, we show that two structural features underlie the mechanism of oligomerisation in Dictyostelium discoideum CAP. Positively charged clusters on the surface of the N-terminal helix-barrel domain are involved in inter-molecular interactions with the N or C-terminal domains. Abolishing these interactions mainly renders dimers due to a domain swap feature in the extreme C-terminal region of the protein that was previously described. Based on earlier studies with yeast CAP, we also generated constructs with mutations in the extreme N-terminal region of Dictyostelium CAP that did not show significantly altered oligomerisation behaviour. Constructs with mutations in the earlier identified protein?protein interaction interface on the N-terminal domain of CAP could not be expressed as soluble protein. Assessment of the soluble proteins indicates that the mutations did not affect their overall fold. Further studies point to the correlation between stability of full-length CAP with its multimerisation behaviour, where oligomer formation leads to a more stable protein.
Keywords: CAP; Srv2; oligomerisation; protein?protein interaction; protein structure
Abbreviations: CAP, cyclase-associated protein; N-CAP, N-terminal domain of CAP; C-CAP, C-terminal domain of CAP; HMW, high molecular weight; SAXS, small angle X-ray scattering; SEC-MALLS, size-exclusion chromatography combined with multiple-angle laser light scattering
Corresponding author.
1 Present addresses: A.M. Yusof, Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA; A. Hofmann, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, QLD 4111, Australia.
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