Journal of Molecular Biology
Volume 362, Issue 5, 6 October 2006, Pages 1072-1081
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Mechanism of Oligomerisation of Cyclase-associated Protein from Dictyostelium discoideum in Solution

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Abstract

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.

Introduction

Cyclase-associated protein (CAP), alias Srv2, is a highly conserved and widely distributed protein required for normal cell growth and development.1 The protein was first identified in Saccharomyces cerevisiae.2 Full-length CAP consists of a N-terminal domain (N-CAP), a proline-rich linker region and a C-terminal domain (C-CAP). In S. cerevisiae, the N-terminal domain is required for Ras response and it binds to the C-terminal region of adenylyl cyclase by coiled-coil interaction.3 The C-terminal domain of CAP binds monomeric actin with a 1:1 molar stoichiometry and inhibits actin polymerisation.4., 5. Meanwhile, the proline-rich middle region of S. cerevisiae CAP is recognized by the Src homology 3 (SH3) domains of several proteins and is also involved in the correct localisation of CAP in the cell.6

Although distinct functions have been allocated to each CAP domain, recent studies revealed that they are not mutually exclusive. Yeast and human CAP have been demonstrated to be involved in recycling actin and cofilin for new rounds of actin depolymerisation and polymerisation.7., 8. Whereas the C-terminal domain of human CAP1 sequesters monomeric actin as expected, the N-terminal domain is able to bind the actin–cofilin complex.7 Thus, it is evident that the functions of the individual CAP domains are intertwined.

Wild-type Dictyostelium CAP is a 50 kDa protein that has about 40% identity to its S. cerevisiae and human counterparts.5 The structure of N-CAP from D. discoideum has been determined by both NMR9 and X-ray crystallography.10 The domain consists of six antiparallel helices arranged into a bundle. In contrast to the N-CAP structures, the C-CAP crystal structures of S. cerevisiae and human revealed the fold of a parallel right-handed β-helix where two molecules form a dimer through the domain-swapping of their extreme C-terminal β-hairpins.11

In our attempts to determine the crystal structure of full-length CAP, we have recently solved the structure of the auto-proteolytic N-CAP fragment of the full-length D. discoideum protein.12 To date, structural studies on full-length CAP have been difficult owing to its tendency to precipitate while being concentrated in the presence of membranes, apart from apparently possessing auto-proteolytic activity.13 Although crystallisation trials were set up with the full-length protein, only N-CAP crystals were obtained, suggesting the occurrence of auto-proteolytic activity in the crystal drop.

N-CAP has been crystallised in three different space groups (P21, P1 and C2221), allowing structural insights into CAP protein–protein interactions from the crystal packing.10., 12. While the monoclinic crystal form contained an N-CAP monomer, the two other forms appeared as dimers. The triclinic and orthogonal crystal forms contained similar, but not identical, side-to-side dimers with a common interface involving the same helices. The orthogonal form also allowed characterisation of a head-to-tail dimer. The presence of two different N-CAP oligomer conformations supports the idea that there are various inter-molecular interactions available to the N-terminal domain.

Information obtained from the N and C-CAP structures support the various functional studies reporting that the full-length protein is able to interact with itself and other CAP molecules to form dimers and multimeric complexes14 (Table 1). The size of yeast CAP oligomers was determined to be in the range of tetramers and dodecamers,17 while Dictyostelium CAP was reported to form hexamers in solution.10 Furthermore, the protein has not been shown to exist as monomers in the cell and its self-association seems to be an important property of CAP. Previous work on cell extracts reported that yeast and mammalian CAP form high molecular weight (HMW) complexes.17., 18. The complexes are inferred to be the interaction between CAP and actin monomers, adenylyl cyclase, other CAP-binding proteins and other CAP molecules.

Intriguingly, to date, there has not been any systematic structural characterisation of full-length CAP in solution, since literature reports have mainly concentrated on either the amino or carboxyl domain. Our ongoing efforts on crystallising the full-length protein have been complicated by its multimerisation behaviour. In order to investigate this behaviour in more detail, we have designed four types of mutants of recombinant His-tagged Dictyostelium CAP, based on the available crystal structures and information from earlier work, to reduce its oligomerisation tendencies. The oligomerisation behaviour of the mutants able to be expressed in solution has been investigated and compared to the wild-type protein. The results indicate that two structural features of the protein are responsible for the mechanism of CAP oligomerisation.

Section snippets

Protein expression and concentration

Not all the Dictyostelium mutants that were generated were expressed in the soluble fraction (Table 2). It is possible that mutants MUT4, 8, 9, 10 and 11 were expressed but are insoluble, suggesting that the amino acid changes affected the folding process. Mutants MUT4 and 8 had residue changes on basic clusters on the surface of N-CAP (Figure 1) while MUT9, 10 and 11 were produced to disrupt the formation of N-CAP side-to-side dimers that were discussed earlier. Nevertheless, since our focus

Two structural features underlie the mechanism of CAP oligomerisation

The main difficulty in working with full-length CAP is its tendency to form HMW aggregates that are clearly visible at the concentration stage of protein purification. CAP multimer formation might very well be a physiological property of the protein and a number of structural features are responsible for this tendency. Crystal structures have revealed the capability of CAP to form domain-swapped dimers with their C-terminal domains and to engage in at least two different types of

Identification of basic surface residues

Surfaces of N-CAP (PDB entry 1TJF) (Figure 1) and C-CAP (PDB entry 1K4Z) crystal structures were inspected with the software GRASP.24 Several basic residues on the surface of N-CAP are clustered and particular surface residues were identified for subsequent mutations inverting the surface charge. The residues chosen were Lys71, Lys72, Lys125, Lys127, Arg131, Lys178, Lys181, Lys203 and Lys206.

Plasmid construction and mutagenesis

Full-length wild-type CAP from D. discoideum was subcloned as described.13 The resulting construct

Acknowledgements

We gratefully acknowledge funding of this study by the BBSRC. We thank Emmajayne Kingham and Anne Helness for their technical help, as well as the Wellcome Trust and Nuffield Foundation for awarding summer studentships to E.K. and A.H., respectively.

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    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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