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Keywords:

  • collapsin response mediator protein 2;
  • crystal structure;
  • growth cone guidance;
  • neuronal regeneration;
  • tetramer;
  • TIM barrel

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the three-dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.

Abbreviations
DHP

dihydropyriminidase

DRP

dihydropyriminidase-related proteins

CRMP

collapsin response mediator protein

MAG

myelin-associated glycoprotein

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TIM

triosephosphate isomerase

Ulip

Unc-33-like proteins

During the development and function of the nervous system, a key process is the coordinated growth of neuronal axons to their targets. Both attractive and repulsive signals are used in order for the neuronal growth cone to find its appropriate target. Semaphorins are a large family of axon guidance proteins. The best characterised one, semaphorin 3 (also called collapsin), is a secreted protein acting as a chemorepellent for axonal growth.

The collapsin response mediator proteins [CRMP; the family is also known as Unc-33-like proteins (Ulip), dihydropyriminidase-related proteins (DRP), TUC (TOAD/Ulip/DRP) and dihydropyriminidase-like proteins] are a family of cytosolic proteins, CRMP-1 to CRMP-5 in humans, related to dihydropyriminidases (DHPs) (Hamajima et al. 1996; Wang and Strittmatter 1996). Out of the CRMP family, CRMP-2 is by far the most extensively characterised member. It has been shown to be O-glycosylated (Cole and Hart 2001) and to have specific functions within the nervous system (see below) (Arimura et al. 2004). The expression of CRMP-2 in the nervous system is developmentally regulated (Kamata et al. 1998; Inagaki et al. 2000). Overexpression of CRMP-2 induces the growth of numerous axons; it is also involved in the maturation of neurites and pre-existing dendrites to axons (Yoshimura et al. 2005). It plays an important role in axonal guidance, being a mediator of semaphorin 3A signalling (Brown et al. 2004). CRMP-2 is phosphorylated by GSK-3 (Cole et al. 2004) and Rho kinase (Arimura et al. 2000; Gu et al. 2000). The phosphorylation by GSK-3 is primed by phosphorylation of CRMP-2 by Cdk5 (Cole et al. 2006).

A specific case of axonal pathfinding is neuronal injury, in which case a major inhibitory factor for growth cone extension is comprised of the proteins of the myelin sheath. CRMP-2 is involved in the regulation of growth cone collapse by myelin-related inhibitors, and its activation can be induced by the binding of the myelin-associated glycoprotein (MAG) on the axonal surface (Mimura et al. 2006). It is also expressed in oligodendrocytes, the myelinating cells of the CNS, in a developmentally regulated manner (Ricard et al. 2000), but its exact functions during myelination remain to be characterised. A number of studies have also suggested a role for CRMP-2 in the aetiology of neurological disorders, including Alzheimer’s disease (Cole et al. 2004; Czech et al. 2004; Kanninen et al. 2004; Sultana et al. 2006). Roles for CRMP-2 have also been suggested in non-neuronal cells (Tahimic et al. 2006).

Collapsin response mediator protein 2 has 58% sequence identity with the human enzyme DHP, which catalyses the second step in pyrimidine degradation; however, no catalytic activity has been observed for any member of the CRMP family, and key active site residues of DHP are not conserved in the CRMPs. It, thus, appears that the CRMPs constitute an example of enzymes that have lost their catalytic function and gained a regulatory role instead. In this respect, it is of interest that CRMP-2 can interact with DHP (Wang and Strittmatter 1997). CRMP-2 has two splice isoforms, differing by the presence of an additional N-terminal sequence in the CRMP-2A isoform. The expression pattern and function of the two isoforms are different (Yuasa-Kawada et al. 2003; Bretin et al. 2005).

Collapsin response mediator protein 2 is able to regulate microtubule dynamics (Gu and Ihara 2000), and it directly binds to tubulin heterodimers (Fukata et al. 2002). The phosphorylation of CRMP-2 by Rho kinase in turn inhibits its binding to tubulin; however, CRMP-2 can bind actin irrespective of its phosphorylation status (Arimura et al. 2005). Rho phosphorylation of CRMP-2 is an event downstream of MAG binding to the neuronal surface (Mimura et al. 2006). CRMP-2 also interacts with Numb, an endocytosis-related protein, possibly indicating a role in the endocytotic recycling of the adhesion molecule L1 at the neuronal growth cone (Nishimura et al. 2003). Attempts to map the binding sites for both tubulin and Numb have been made using deletion experiments (Fukata et al. 2002; Nishimura et al. 2003). The phosphorylation sites, and most likely also the glycosylation sites, lie within the disordered C terminus of the protein.

We have determined the crystal structure of human CRMP-2. The structure is used to analyse differences between members of the CRMP family and other homologous proteins. Furthermore, existing data on CRMP-2 and its interactions are discussed based on the structure. A plausible model, based on the crystal structures of CRMP-1 and CRMP-2, for a heterotetramer between CRMP-1 and CRMP-2 is also presented.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cloning and expression

The coding sequence for human CRMP-2 (residues 13–490), excluding the C-terminal region predicted to be disordered, was subcloned into the expression vector pNIC-Bsa4, resulting in a construct with an N-terminal hexahistidine tag and an integrated TEV protease cleavage site. The plasmid was transformed into Escherichia coli BL21(DE3) cells. For expression, cells were grown in 1500 mL Terrific Broth (containing 7% glycerol, 50 μg/mL kanamycin and 100 μL BREOX) in bubble flasks in the large scale expression system (developed at Structural Genomics Consortium, Toronto).

Cells were grown at +37°C until an optical density of 2.5 at 600 nm was reached. The cultures were down-tempered to +18°C for 1 h in a water bath. The expression of CRMP-2 was induced by the addition of 0.5 mmol/L IPTG, and expression was allowed to continue overnight at +18°C.

Cells were harvested by centrifugation, and the pellets were suspended in IMAC buffer 1 (50 mmol/L sodium phosphate pH 7.5, 500 mmol/L NaCl, 10% glycerol), supplemented with Complete EDTA-free protease inhibitors (Roche, Basel, Switzerland), and frozen at −80°C until used.

The cells were briefly thawn in warm water, and 2000 U of benzonase were added. The disruption of the cells was performed by three rounds of high-pressure homogenisation at 10 000 PSI, the samples were centrifuged for 20 min at 40 000 g, and the soluble fraction was filtered.

Collapsin response mediator protein 2 purification

Purification was conducted automatically on an ÄKTA Xpress system at a flow of 0.8 mL/min. Prior to purification, the HisTrap HP and Superdex 200 columns were equilibrated with IMAC buffer 1 (50 mmol/L HEPES pH 7.5, 10 mmol/L imidazole, 500 mmol/L NaCl, 10% glycerol, 0.5 mmol/L TCEP) and gel filtration buffer (20 mmol/L HEPES pH 7.5, 300 mmol/L NaCl, 10% glycerol, 0.5 mmol/L TCEP), respectively. The protein sample was loaded on the HisTrap HP column that was washed with IMAC buffer 1 followed by IMAC buffer 2 (50 mmol/L HEPES pH 7.5, 50 mmol/L imidazole, 500 mmol/L NaCl, 10% glycerol, 0.5 mmol/L TCEP). Bound protein was eluted from the IMAC columns with 7.5 mL of IMAC elution buffer (50 mmol/L HEPES pH 7.5, 400 mmol/L imidazole, 500 mmol/L NaCl, 10% glycerol, 0.5 mmol/L TCEP) and loaded onto the gel filtration column. The chromatogram from gel filtration showed one major protein peak that consisted of highly pure CRMP-2. TCEP was added to the pooled protein peak to a final concentration of 2 mmol/L. The protein was concentrated to 33 mg/mL and stored at −80°C.

Crystallisation and data collection

Collapsin response mediator protein 2 was crystallised with the hanging drop method at +20°C, by mixing 2 μL of the well solution (0.2 mol/L CaCl2, 0.1 mol/L Tris pH 8.5, 14–18% PEG10000) and 1 μL of protein (17 mg/mL) and equilibrating against 0.5 mL of the well solution.

A crystal was briefly soaked in a cryoprotectant solution (15% glycerol, 0.1 mol/L Tris pH 8.5, 18% PEG 10000, 0.2 mol/L CaCl2, 0.3 mol/L NaCl, 20 mmol/L HEPES pH 7.5, 2 mmol/L TCEP) prior to data collection. X-ray diffraction data were collected at 100 K on the ESRF beamline ID14.2 (Grenoble, France). The data were processed using XDS (Kabsch 1993) and XDSi (Kursula 2004), and 5% of the data were randomly selected for the calculation of the free R factor. The data processing statistics are shown in Table 1.

Table 1.   Data collection and structure refinement. The values in parentheses correspond to the high-resolution shell
Space groupP21
Unit cell (a,b,c,β)86.4 Å, 126.1 Å, 102.9 Å, 113°
Resolution (Å)30–2.4 (2.6–2.4)
Optical resolution (Å)1.76
Rsym (%)14.6 (46.6)
<I/σI>9.4 (3.3)
Completeness (%)99.7 (99.7)
Redundancy4.3 (4.3)
Rcryst (%)17.0
Rfree (%)24.5
r.m.s. deviation bond length (Å)0.015
r.m.s. deviation bond angle (°)1.6
Average B factors (Å2)
Protein (A,B,C,D)23,23,23,22
Solvent22
Calcium ions35

Structure solution and refinement

The structure was solved using molecular replacement in Molrep (Vagin and Teplyakov 1997) with a monomer from the mouse CRMP-1 structure (PDB entry 1KCX) (Deo et al. 2004) as template, and four monomers of CRMP-2 were found within the asymmetric unit, arranged into a tetrameric assembly. The structure was refined with data between 30 and 2.4 Å using Refmac (Murshudov et al. 1997), employing loose non-crystallographic symmetry restraints between the four monomers, and iterative model building between refinement rounds was carried out in coot (Emsley and Cowtan 2004). Water molecules were added into difference electron density maps using ARP/wARP (Perrakis et al. 1999). In the final model, 87.7% of all residues were in the most favoured region of the Ramachandran plot, and no residues were in the disallowed regions. Proline residues 305 and 396 were modelled as cis-prolines. The final refinement statistics are summarised in Table 1. The coordinates and structure factors were deposited in the Protein Data Bank under the accession code 2GSE.

The structure was analysed using the programs ProFunc (Laskowski et al. 2005a), PDBsum (Laskowski et al. 2005b), PQS (Henrick and Thornton 1998), ElNemo (Suhre and Sanejouand 2004) and SSM (Krissinel and Henrick 2004). Figures were made using Dino, POV-ray, POVscript+ (Fenn et al. 2003), ESPript (Gouet et al. 1999), APBS (Baker et al. 2001), PyMol and UCSF Chimera (Pettersen et al. 2004).

Covalent cross-linking

The oligomeric status of purified CRMP-2 was analysed by covalent cross-linking and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In brief, 1.3 mg/mL CRMP-2 was incubated at 22°C for 15 min in 100 mmol/L HEPES (pH 7.3) containing either 50, 150 or 500 mmol/L NaCl. Then, glutaraldehyde was added to a final concentration of 10, 25 or 100 mmol/L, and incubation was continued at 22°C for 30 min. The reaction was stopped by the addition of SDS–PAGE sample buffer, and the samples were analysed by electrophoresis.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The overall structure of human collapsin response mediator protein 2

The crystal structure of human CRMP-2 was determined at 2.4-Å resolution. The C-terminus of the CRMPs has been shown to be cleaved off by partial proteolysis (Deo et al. 2004), and thus, this region was not included in the expression construct. The largest fraction of the monomer structure is folded into a triosephosphate isomerase (TIM) barrel; another well-defined domain is the small β-sheet domain formed by the N-terminal residues and segments close to the C terminus of the folded CRMP-2 (Fig. 1). As expected from sequence homology, the fold of CRMP-2 is closely related to that of the enzymes related to DHPs (Table 2). They all have in common the TIM-barrel domain and the small domain. Normal mode analysis (Suhre and Sanejouand 2004) of the structure indicates that it is likely that the small β-sheet domain of CRMP-2 is flexible with respect to the rest of the monomer (not shown). As the active site in the related DHPs lies on the other side of the TIM barrel, there is little clue as to the function of the small domain. Most probably it is involved in protein–protein interactions (see below).

image

Figure 1.  The crystal structure of human collapsin response mediator protein 2 (CRMP-2). (a) Folding of the monomer. The small domain is shown in yellow, the TIM barrel in green/red, and the C-terminal helix in magenta. (b) The electrostatic potential mapped onto the surface of one monomer. The positively charged region corresponds to the C-terminal helix. (c) A CRMP-2 tetramer as seen in the crystal structure. The view on the right has been rotated 90 degrees with respect to the one on the left. The locations of the tetramerisation interfaces 1 and 2, as discussed in the text, are shown by arrows. (d) The phosphorylation target Ser465. The molecular surface of the CRMP-2 monomer is shown, and indicated are the positions of Ser465 and two nearby basic residues (blue). The darker gray shading indicates the location of the small domain.

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Table 2.   Superpositions of human collapsin response mediator protein 2 (CRMP-2) with selected homologous proteins. Monomer A of CRMP-2 was used in the program SSM
ProteinPDB code/referencesr.m.s. deviation (# residues)
CRMP-1 (Mus musculus)1KCX (Deo et al. 2004)0.72 (475)
DHP (Saccharomyces kluyveri)2FVK (Lohkamp et al. 2006)1.70 (440)
DHP (Dictyostelium discoideum)2FTW (Lohkamp et al. 2006)0.76 (463)
Urease α subunit (Bacillus pasteurii)1IE7 (Benini et al. 2001)2.46 (309)
Isoaspartyl dipeptidase (Escherichia coli)1ONW (Thoden et al. 2003)2.38 (308)

The asymmetric unit consists of a homotetramer with nearly 222 symmetry (Fig. 1c). In the tetramer, the total buried solvent-accessible surface area is over 9400 Å2, heavily suggesting the presence of a physiologically important tetramer. A superposition of the four monomers on each other gives root mean square (r.m.s.) deviations for the Cα positions of 0.2–0.3 Å between the different monomers, indicating strong similarity between the monomers in the asymmetric unit. In line with the homotetrameric structure, common to the structural family, the majority of CRMP-2 eluted from gel filtration as a broad peak with a molecular weight of 150 kDa, corresponding to a trimer, with a minor fraction of monomer detectable (Fig. 2). The result is reproducibile between different batches of pure CRMP-2 protein as well as between protein constructs differing in length by a few amino acids (unpublished data). All peaks from the gel filtration consisted of CRMP-2, which is a clear indication of the presence of various homo-oligomeric states (Fig. 2b). It is possible that the difference from the expected tetramer molecular weight reflects a rapid dynamic equilibrium between dimeric and tetrameric states of CRMP-2, also supported by the width of the peak covering both the dimeric and tetrameric molecular weights. One of the best-characterised such equilibria is that seen in haemoglobin; in fact, gel filtration has been used to perform detailed studies on haemoglobin dimer-tetramer kinetics, the observed peak corresponding roughly to the size of a trimer (Manning et al. 1996). Furthermore, a trimer cannot be rationally explained by our structure, which clearly is a ‘dimer of dimers’. It is possible that the equilibrium results from different oligomerisation kinetics between the full-length CRMP-2 and our truncated version, lacking the C-terminal unstructured tail. In addition, a minor peak corresponding to a size of an octamer was observed. In covalent cross-linking (Fig. 2c), the main species of CRMP-2 that were observed corresponded to monomers, dimers and tetramers.

image

Figure 2.  Purification and oligomeric state of human collapsin response mediator protein 2 (CRMP-2). The theoretical molecular weight of the expressed protein is 55 kDa. (a) The elution volume in size exclusion chromatography indicates the presence of oligomers, probably a mixture of dimers and tetramers. The elution volumes of molecular weight markers (in kDa) from the same column are indicated above the graph. (b) SDS–PAGE analysis of the fractions confirms that all observed peaks consist of pure human CRMP-2. Samples: 1- molecular weight marker (sizes indicated in kDa on the left), 2- fraction D10 (octamer), 3-fraction D8, 4- fraction D6, 5- fraction D4, 6- fraction D2, 7- fraction E1 and 8- fraction E3 (monomer). (c) Covalent cross-linking of purified CRMP-2. Glutaraldehyde concentrations: 1–3 : 10 mmol/L; 4–6 : 25 mmol/L; 7–9 : 100 mmol/L. NaCl concentrations: 1,4,7 : 50 mmol/L; 2,5,8 : 150 mmol/L; 3,6,9 : 500 mmol/L. The positions of monomer (*), dimer (**) and tetramer (****) are indicated on the right, the positions of molecular weight markers (kDa) are on the left. At the highest salt concentration, CRMP-2 is cross-linked into high-molecular weight aggregates that do not enter the gel. Increasing the amount of cross-linker at the lower salt concentrations clearly increases the amount of cross-linked tetramer (compare lanes 1, 4 and 7).

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Recently, Ser465 was identified as a CRMP-2 phosphorylation site in mouse brain (Vosseller et al. 2005). This is the only characterised phosphorylation site lying within the structure determined here, i.e. outside the C-terminal tail. Ser465 is easily accessible in the tetramer, lying in an exposed loop leading to the last helix of each monomer. The phosphorylation of serine 465 is unique to CRMP-2, as this residue is not conserved in the other four CRMP proteins. CRMP-5 has a threonine in this position that possibly could be phosphorylated. In human DHP, the corresponding residue is an aspartic acid. Nearby Ser465, two basic residues are found, one from the same loop and one from the small domain, which could interact with the phosphorylated residue (Fig. 1d). This could, for example, generate a stabilising salt bridge between the small domain and the rest of the CRMP-2 molecule. It is also possible that Ser465 phosphorylation could affect interactions between the folded core of the protein and the positively charged C-terminal tail.

Comparison of CRMP-1 and -2 and heterotetramerisation

The structure of murine CRMP-1 was solved recently (Deo et al. 2004), thus permitting a detailed structural comparison between these two closely related proteins. Despite the high overall similarity of the structures (Fig. 3), differences may also be found that could have functional implications.

imageimage

Figure 3.  Superposition of human collapsin response mediator protein 2 (CRMP-2) with mouse CRMP-1. (a) The A monomer from each structure was superimposed. Cyan-CRMP-2, gray-CRMP-1. (b) Tetramerisation. The superposition is based on the A monomers to highlight differences in subunit orientations. CRMP-2: A-cyan, B-green, C-orange, D-magenta. CRMP-1 is shown in grey. (c) A sequence alignment between human and mouse CRMP-1, human CRMP-2, and human DHP. The secondary structure elements are as seen in our structure of human CRMP-2. The secondary structures forming the TIM barrel are shown in grey, and the numbering of the elements with respect to the TIM barrel structure is shown in parentheses. Note how the homology between DHP and the CRMPs extends to the region of the last helix.

Collapsin response mediator protein 1 and CRMP-2 are able to form heterotetramers with each other (Wang and Strittmatter 1997), and we, thus, compared the monomer–monomer interfaces within the CRMP-1 and CRMP-2 homotetramers to shed further light on these interactions (Fig. 4). This analysis was, to some extent, carried out based on the structure of CRMP-1 and a sequence alignment for CRMP-2 (Deo et al. 2004), but having both experimentally determined structures at hand, we bring novel aspects to this comparison.

image

Figure 4.  The tetramerisation interfaces in human collapsin response mediator protein 2 (CRMP-2). Differences in the oligomerisation interfaces between CRMP-1 and CRMP-2 are also pointed out. (a) A central region of interface 1, with three sequence differences. In CRMP-2, Gln266 forms part of a hydrophilic interface, while in CRMP-1, Leu266 points away from interface 1. (b) The interaction of the C-terminal helix with the opposing monomer, also part of interface 1. The main difference concerns Lys480 of CRMP-2 (glutamine in CRMP-1). Arg485 interacts with backbone carbonyl groups from both monomers, having no negative counter-charge nearby. (c) Features of interface 2 and main differences between CRMP-1 and CRMP-2. In the centre of the interface, residue 237 is not conserved, and the relative properties of the sequence differences in the vicinity result in a much more hydrophobic interface for CRMP-1. At the edge of the hydrophobic patch, CRMP-1 has an intersubunit salt bridge, which is missing in CRMP-2.

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Interface 1 is formed between monomers A–B and C–D, generating a dimer with a dyad axis. Most of the residues in this interface are conserved between CRMP-1 and CRMP-2, indicating that this interface probably can be used to make hetero-oligomers. The differences in this region between CRMP-1 and CRMP-2 are shown in Fig. 4a. The most significant differences concern residues 266 and 316, Leu and Tyr in CRMP-1 and Gln and Phe in CRMP-2, respectively.

Interface 2, between the two dimers related by interface 1, is also required for tetramer formation. Interestingly, several of the non-conserved residues between CRMP-1 and CRMP-2 are found at this interface (Fig. 4c). This is likely to adversely affect hetero-oligomerisation via interface 2. This interface is also much more hydrophobic in CRMP-1 than in CRMP-2. For example, in CRMP-1, Phe237 from monomers A and C face each other at a distance of 3.3 Å; in CRMP-2, the corresponding residue is Asn237, being 3.5 Å away from Asn237 on the opposing monomer. In addition, the above-mentioned Leu266 in CRMP-1 contributes to both interfaces 1 and 2, but Gln266 in CRMP-2 points towards interface 1. From the differences, it would seem logical that a CRMP-1/CRMP-1 interaction at this interface should be stronger than a CRMP-1/CRMP-2 interaction (or a CRMP-2/CRMP-2 interaction).

The pocket corresponding to the active site region of DHP

The active sites of DHP and related enzymes lie within the TIM barrel, such that the cavity opens towards the C-terminal end of the barrel. In CRMP-1 and CRMP-2, a cavity also exists at this position. The volume of this cavity in CRMP-2 is 1400 Å3 and the depth 16 Å. Catalytic residues of DHP have not been conserved in CRMP-2; for example, the catalytic lysine and two of the Zn-coordinating histidine residues are not present (Fig. 5). However, it is possible that this site can be used to bind small molecules; an indication of the presence of a ligand-binding potential in the cavity is the presence of a well-defined Ca2+ ion in CRMP-2 in the cavity. On the other hand, one of the most divergent regions between CRMP-1 and CRMP-2 is formed by the walls and immediate surroundings of this cavity, which may either indicate no evolutionary pressure for conservation or the formation of different binding cavities in the different CRMPs. For example, CRMP-1 has three tyrosine residues in the walls of this pocket, none of which are present in CRMP-2 (Fig. 5b).

image

Figure 5.  The active site region of DHP in the collapsin response mediator proteins (CRMPs). (a) A superposition of CRMP-2 (cyan) and DHP (orange) from Saccharomyces kluyveri (PDB entry 2FVK) (Lohkamp et al. 2006). In the centre, the DHP substrate (dihydropyrimidine-2,4(1H,3H)-dione) and two zinc ions (green) are seen. The residue numbering refers to that of CRMP-2, and the corresponding residue in DHP is indicated in parentheses, if different. Note the substitution of the catalytic lysine and two of the zinc-coordinating histidines in CRMP-2. (b) The view is into the pocket from the C-terminal end of the TIM barrel. CRMP-2 is shown in cyan and CRMP-1 in grey colours. The residues lining the pocket are thin for conserved residues and thick for residues that are different in CRMP-1 and CRMP-2. The calcium ion in CRMP-2 is shown as a magenta sphere.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We have determined the first crystal structure of human CRMP-2, by far the best characterised member of the CRMP family, with roles in neurological development and disease. CRMP-2 is a homotetramer with high structural homology to CRMP-1 and the DHPs. Based on the analysis of the tetramer interfaces in the crystal structures of CRMP-1 and CRMP-2 homotetramers, we are able to propose a model for CRMP-1/2 heterotetramers, where two CRMP-1 and two CRMP-2 subunits arrange into homodimers via interface 2. These homodimers then form a heterotetramer via interface 1. The model for the heterotetramer is presented in Fig. 6.

image

Figure 6.  A proposed model for the collapsin response mediator protein (CRMP-1/2) heterotetramer, based on the structural comparison. CRMP-1 monomers are indicated in yellow/orange colours, while CRMP-2 molecules are light and dark blue.

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As is the case with CRMP-1 (Deo et al. 2004), the tetramerisation of CRMP-2 is similar to that of DHP, apart from minor conformational differences e.g. in the small domain and the presence of the C-terminal helix that is also involved in oligomerisation. This C-terminal helix is the only region on the CRMP-2 surface with a positive charge potential (Fig. 1b). Regarding DHP tetramerisation, it is important to note that in fact, the human DHP sequence is highly homologous to that of CRMP-1 and CRMP-2 (Fig. 3c) also in the region of the C-terminal helix, and residues involved in tetramerisation in this region are conserved. Thus, human DHP most likely also has a C-terminal helix such as that seen in the CRMPs, and fine details of tetramerisation probably more closely resemble those of the CRMPs than those of previously determined DHP structures from yeasts and bacteria. CRMP-2 is also able to interact directly with DHP (Wang and Strittmatter 1997), and it is likely that CRMP-2 is able to regulate DHP activity via hetero-oligomer formation with active DHP.

The pocket corresponding to the active site of DHP is also present in CRMP-2, within the TIM barrel. No small molecule ligands for CRMP-2 have been reported, but this pocket, which is rather different in CRMP-1 and CRMP-2, could be used to bind such ligands. Whether calcium, which was required to obtain CRMP-2 crystals (data not shown) and was found in this pocket, could be a physiological ligand for CRMP-2, needs to be determined. It is likely that the binding is non-specific, taking into account both the high experimental Ca2+ concentration and the fact that only a single calcium-coordinating oxygen comes from the CRMP-2 protein. We plan to further study the ligand-binding properties of CRMP-2, with a specific emphasis on this pocket.

N- and C-terminal domains of CRMP-2

The mutation of the loop on the top of the small domain (residues 46–57) to polyalanine in CRMP-1 was shown to generate a constitutively active form of the protein (Deo et al. 2004). The sequence of this region is identical in CRMP-2. Comparing the loop in CRMP-1 and CRMP-2 indicates a slightly different conformation, and overall, it can be concluded that the loop is not tightly fixed onto the top of the small domain; for example, Leu49 has limited hydrophobic interactions despite its pointing into the protein (Fig. 7a). In addition, in our structure, all side chains of the loop were defined in electron density and could be modelled, leading to a more accurate model of this functionally important region. In the homologous urease α subunit, this loop is much longer and interacts closely with the β subunit (Benini et al. 2001). Thus, this loop may be a conserved site for protein interactions in this structural family.

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Figure 7.  Previously mapped interaction domains of collapsin response mediator protein 2 (CRMP-2) in the structure. (a) The loop (residues 46–57) in the small N-terminal domain is well defined in the electron density. Cyan-CRMP-2, grey-CRMP-1. The electron density is that of the final 2Fo–Fc map, contoured a 1 σ. For CRMP-1, most side chains are not present in the model, and for example Leu49 was modelled to point outwards from the loop. In CRMP-2, Leu49 is well-defined, together with the rest of the loop. (b) Overall view with coloured mapped domains for protein interactions, such as determined mainly by using truncation mutants in previous studies. Note how the previously suggested minimal binding sites for both Numb (275–322, blue) and tubulin (323–381, red) are buried within the CRMP-2 fold. The loop on top of the small domain (residues 46–57) is shown in orange.

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Alternative splicing has been reported to generate two isoforms of CRMPs 1–4, differing in their N-terminal sequences (Yuasa-Kawada et al. 2003). The structure presented here corresponds to the shorter, better characterised, isoform, occasionally called CRMP-2B in the literature. Our results, thus, confirm that the folded region which is comprised of the small domain and the TIM barrel domain (residues 1–489) is enough for tetramer formation, and thus, neither the extended N-terminus for the alternatively spliced forms nor the unfolded C-terminal region is required for oligomerisation. The extra sequence present in the longer form, CRMP-2A, bears no significant similarity to proteins outside the CRMP family, apart from a slightly extended homology to the urease α subunit (data not shown). Interestingly, the two splice isoforms of CRMP-2 have opposite effects on microtubule organisation and axonal growth, indicating that the N-terminal extension is functionally important in regulating CRMP-2 activity (Yuasa-Kawada et al. 2003). Furthermore, only CRMP-2B is expressed by oligodendrocytes, cells forming the myelin sheath in the CNS, while both CRMP-2 isoforms are present in axons (Bretin et al. 2005). Proteolytic cleavage of the N-terminus in CRMP-2A also occurs after the activation of NMDA receptors (Bretin et al. 2006).

The C-terminal ‘tail’ region of the CRMPs has no detectable sequence homology to other proteins, it has a high pI of 10.8 and a high content of serine residues and it is predicted to be unfolded in secondary structure predictions (not shown). These attributes resemble those usually associated with the tau/MAP microtubule-binding proteins. In a recent study, a C-terminally truncated form of CRMP-2 was shown to be specific to the PNS; the expression of this form was decreased after sciatic nerve injury (Katano et al. 2006). It has also been shown that the CRMP C-terminal tail is cleaved off by proteolysis both in vitro and in vivo (Deo et al. 2004; Bretin et al. 2006), and it was, thus, not included in our construct. However, it is worth mentioning that important functional sites reside in this domain.

Collapsin response mediator protein 2 is regulated by phosphorylation by a number of kinases, and all phosphorylation sites, with one exception, Ser465, have been mapped to the C-terminal tail (Arimura et al. 2000; Gu et al. 2000; Brown et al. 2004; Cole et al. 2004; Yoshimura et al. 2005). Phosphorylation inhibits the binding of CRMP-2 to tubulin and prevents its effect on tubulin polymerisation (Arimura et al. 2005). It is likely that the C-terminal tail plays a major role in interactions between CRMP-2 and tubulin. Furthermore, the interaction between CRMP-2 and the tetratricopeptide repeat (TPR) domain of kinesin seems to be mediated by residues 440–572, i.e. the C-terminal tail (Kimura et al. 2005).

Collapsin response mediator protein 2 is also O-glycosylated (Cole and Hart 2001; Kanninen et al. 2004), and prediction of glycosylation sites using the NetOGlyc server (Hansen et al. 1998) strongly suggests that the glycosylation site(s) also are in the C-terminal tail (not shown). The effects of glycosylation on CRMP-2 function have not been defined, but it is possible that glycosylation regulates the interactions of CRMP-2 with its ligands and/or its phosphorylation.

Interestingly, a recent screen for brain calmodulin-binding proteins identified CRMP-2 as a putative CaM target (Zhang et al. 2006). Searching for putative CaM-binding sequences (Yap et al. 2000) of CRMP-2, the most likely region would be the last helix of the current structure, residues 475–489. The helix is involved in the tetramerisation interface, not being completely buried, however. Another putative CaM-binding region could be the C-terminal tail. While further studies on CRMP-2–CaM interactions are clearly required, it can be speculated that CaM binding could affect CRMP-2 tetramerisation, its interactions with other proteins, and/or its post-translational modifications.

Previously mapped functional domains and their relation to the three-dimensional structure of CRMP-2

A number of studies have been carried out to find interaction partners for CRMP-2. These studies have, to a large extent, used truncation mutants in cell culture experiments, which have also allowed the mapping of binding domains (Fig. 7b). Apart from a short region around residue 320, none of the studied segments are involved in tetramerisation interfaces. The interaction data can now be discussed in light of the current structural information.

Collapsin response mediator protein 2 binds tubulin and regulates microtubule assembly (Gu and Ihara 2000; Fukata et al. 2002). The functional domain in this case was mapped to the region 323–381, based on a series of truncation mutants. This region was the most effective in promoting microtubule assembly in vitro; it was not enough for in vivo function, however (Fukata et al. 2002). Judging from our structure (Fig. 7b), this region is mostly buried within the fold of CRMP-2 (indeed forming an α/β unit of the TIM barrel), and it is likely that the effects observed with either only this domain present or with CRMP-2 lacking this region, are affected by folding defects.

Collapsin response mediator protein 2 also interacts with Numb, a protein involved in endocytosis (Nishimura et al. 2003). It was suggested that the region 275–322 is required for the binding, based on the behaviour of various truncation mutants. On the basis of the crystal structure, the inactive 1–275 mutant used in the interaction study is most likely misfolded, as it contains a truncated TIM domain, and the recorded lack of activity should therefore be interpreted with caution. The active 1–381 mutant, however, contains the entire TIM barrel and most of the small domain. A direct interaction has also been characterised between CRMP-2 and phospholipase D2, requiring residues 243–300 of CRMP-2 (Lee et al. 2002). This region overlaps with that suggested to bind Numb, and forms a central part of the folded TIM barrel domain.

In summary, while the actual interactions of full-length CRMP-2 are not brought into question by the crystal structure of human CRMP-2 presented in this article, the detailed mapping data from truncation experiments are very difficult to rationalise while analysing the three-dimensional structure. This is, of course, a general problem while mapping binding sites by using linear truncation mutants, when the three-dimensional structure of the studied protein is not accurately known. Based on our work, structure-based experiments can now be carried out on CRMP-2 and its interaction properties.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The Structural Genomics Consortium is a registered charity (number 1097737) funded by VINNOVA, the Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research, the Karolinska Institute, the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, and the Canadian Foundation for Innovation. The work was also supported by the Swedish Cancer Society and the Swedish Research Council (PN). PK is an Academy Research Fellow (Academy of Finland).

References

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  3. Materials and methods
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  5. Discussion
  6. Acknowledgements
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