Journal of Molecular Biology
Mechanism of Oligomerisation of Cyclase-associated Protein from Dictyostelium discoideum in Solution
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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Cited by (8)
Native cyclase-associated protein and actin from Xenopus laevis oocytes form a unique 4:4 complex with a tripartite structure
2021, Journal of Biological ChemistryCitation Excerpt :We hypothesize that MGD corresponds to a tetramer of the HFD of XCAP1 and that each arm domain in the high state (Arm-HS) corresponds to a heterotetramer containing a dimer of the CARP domain of XCAP1 and two G-actin molecules (Fig. 6). The HFD of CAP by itself forms a dimer (12, 26, 27), and the N-terminal oligomerization motif forms a putative coiled-coil and mediates formation of a tetramer (34) or hexamer (10, 24, 25) of the HFD. The height of MGD (Figs. 2C, 3F and 4F) matches with that of the diameter of one HFD (12, 26), suggesting that each HFD is laterally attached to the substrate.
Structure and function of a G-actin sequestering protein with a vital role in malaria oocyst development inside the mosquito vector
2010, Journal of Biological ChemistryCitation Excerpt :We show that the sequence PEQ(F/Y) before the penultimate β-strand of the C-CAP fold is indicative for dimer formation (Fig. 4B). Incidentally, the absence in yeast CAP/Srv2p of a small aromatic side chain (Phe or Tyr), which in our structure stack to each other at the dimer interface, may be compensated for by other dimer interactions, such as those of the yeast N-terminal CAP domain (9, 16). The internal Cys/Ser side chain stack is shared with other β-helical proteins (41), but a prominent difference between the parasite and yeast C-CAP structures is the dipartite ladder observed in our CpC-CAP structure, with an N-terminal Cys/Ser stack and a C-terminal hydrophobic stack (supplemental Fig. S2).
CAPt’n of Actin Dynamics: Recent Advances in the Molecular, Developmental and Physiological Functions of Cyclase-Associated Protein (CAP)
2020, Frontiers in Cell and Developmental BiologyPhosphorylation of the cytoskeletal protein CAP1 controls its association with cofilin and actin
2014, Journal of Cell Science
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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.