Crystal and solution structure, stability and post-translational modifications of collapsin response mediator protein Viivi Majava1, Noora Loytynoja1, Wei-Qiang Chen2, Gert Lubec2 and Petri Kursula1 ă Department of Biochemistry, University of Oulu, Finland Department of Pediatrics, Medical University of Vienna, Austria Keywords divalent cations; nervous system; oligomeric status; protein structure; small-angle X-ray scattering Correspondence P Kursula, Department of Biochemistry, University of Oulu, P.O Box 3000, FIN-90014 Oulu, Finland Fax: +358 5531141 Tel: +358 44 5658288 E-mail: petri.kursula@oulu.fi Database The coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 2VM8 (Received 28 April 2008, revised 14 July 2008, accepted 17 July 2008) The collapsin response mediator protein (CRMP-2) is a central molecule regulating axonal growth cone guidance It interacts with the cytoskeleton and mediates signals related to myelin-induced axonal growth inhibition CRMP-2 has also been characterized as a constituent of neurofibrillary tangles in Alzheimer’s disease CD spectroscopy and thermal stability assays using the Thermofluor method indicated that Ca2+ and Mg2+ affect the stability of CRMP-2 and prevent the formation of b-aggregates upon heating Gel filtration showed that the presence of Ca2+ or Mg2+ promoted the formation of CRMP-2 homotetramers, and this was further proven by small-angle X-ray scattering experiments, where a 3D solution structure for CRMP-2 was obtained Previously, we described a crystal structure of human CRMP-2 complexed with calcium In the present study, ˚ we determined the structure of CRMP-2 in the absence of calcium at 1.9 A 2+ was omitted, crystals could only be grown in the resolution When Ca presence of Mg2+ ions By a proteomic approach, we further identified a number of post-translational modifications in CRMP-2 from rat brain hippocampus and mapped them onto the crystal structure doi:10.1111/j.1742-4658.2008.06601.x Axonal growth cone guidance is a tightly regulated process central to both nervous system development and its repair after injury One of the proteins shown to play a specific role in growth cone guidance is the collapsin response mediator protein (CRMP-2) [1–3], also known as dihydropyriminidase-related protein (DRP-2, DPYSL-2), unc-33 like protein (Ulip-2) and turned on after division 64 kDa (TOAD-64) CRMP-2 is a member of the family of collapsin response mediator proteins, which is comprised of five related proteins in humans [4] The crystal structure of human CRMP-2 has previously been determined to a ˚ resolution of 2.4 A [5] from crystals grown in the presence of calcium ions Structurally, CRMP-2 is homologous to the dihydropyriminidases (DHP), with a major part of its 3D structure being formed as a (ba)8 barrel, but it has no characterized catalytic activity of its own, nor have any specific small-molecule ligands for CRMP-2 been identified However, interactions between CRMP-2 and other proteins, such as tubulin [6], Sra-1 [7], Numb [8], a2-chimaerin [9] and phospholipase D [10], have been described CRMP-2 is a homotetramer, but it can also form heterotetramers with other members of the CRMP ⁄ DHP structural family [11] It is possible that its functional mechanism in neuronal development relates, at least partly, to its ability to bind to and regulate other homologous proteins in the family Recently, CRMP-2 has been highlighted as a target for drug development against nervous system disorders, Abbreviations CRMP-2, collapsin response mediator protein 2; DHP, dihydropyriminidase; SAXS, small-angle X-ray scattering FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4583 Structural properties of CRMP-2 V Majava et al such as epilepsy [12], Alzheimer’s disease and nerve injury [3] Its association with depression and schizophrenia has also been studied [13,14] CRMP-2 also forms a part of the signal transduction cascade related to axonal growth inhibition brought about by myelin components, such as the myelin-associated glycoprotein [15], a mechanism that prevents the regeneration of myelinated axons in the central nervous system Non-neuronal expression for CRMP-2 has also been reported [16] and, recently, it has been identified as a putative marker for colorectal carcinoma [17] The best characterized function for CRMP-2 relates to its interactions with cytoskeletal components, especially tubulin [6,18] CRMP-2 is able to regulate the formation of microtubules and, accordingly, it is highly concentrated in growing axons CRMP-2 binds to tubulin heterodimers, and its overexpression in neurons promotes axonal growth and branching [6] Extensive post-translational modifications have been detected for CRMP-2 [19–25]; mainly, this has concerned phosphorylation of the C-terminal tail, which is predicted to be unfolded In addition, CRMP-2 has been characterized as a major target for oxidation in the aging brain [23,26–29] Changes in CRMP-2 posttranslational modifications have also been suggested to play a role in Alzheimer’s disease [19–21,26–29] In the present study, we describe the crystal structure of human CRMP-2 in the absence of calcium Instead of calcium, magnesium ions were required to ˚ grow the crystals, resulting in a 1.9 A structure of CRMP-2 being obtained The effects of Ca2+ and Mg2+ on the CRMP-2 structure were also analysed by CD spectroscopy, the Thermofluor method, smallangle X-ray scattering (SAXS) and gel filtration At a 20 mm concentration, both CaCl2 and MgCl2 stabilize the protein and promote the formation of tetramers The structure was further analysed by mapping posttranslational modifications, as detected using advanced proteomics methods [30], onto the 3D structure of the folded core domain of CRMP-2 Results CD spectroscopic analysis of CRMP-2 conformation and stability in solution To characterize the effects of divalent cations on CRMP-2 structure and stability, CD spectroscopy was carried out in the presence of NaCl, CaCl2 and MgCl2 The CD spectra of CRMP-2 in all tested conditions were similar, indicating the expected presence of both a and b secondary structures However, in the presence of Ca2+ and Mg2+, the CD signal was significantly 4584 stronger, suggesting a higher average content of secondary structure in solution NaCl had no effect on the CD spectrum (Fig 1A) Temperature scans of the samples revealed an intriguing phenomenon upon denaturation In buffer alone, CRMP-2 underwent a structural transition at approximately 50 °C, which resulted in an increase in ellipticity at 220 nm (Fig 1B); this is opposite to the effect generally expected upon protein denaturation The ellipticity did not significantly decrease, even at temperatures approaching 100 °C (data not shown) A CD spectrum recorded at 90 °C shows that the transition involved a complete loss of helical structure and a significant increase in the amount of b structure (Fig 1C) After cooling down, the spectrum at room temperature indicated that the observed structural transition into a b-aggregate was irreversible (data not shown) A sample analysed in the presence of 50 mm NaCl behaved essentially the same (data not shown) In the presence of Ca2+ or Mg2+, however, heat denaturation proceeded as expected, with a sharp decrease in ellipticity at 220 nm at the melting temperature and complete loss of secondary structure (Fig 1B,C) Increasing the concentration of these ions from 20 to 200 mm resulted in a decrease by several degrees in the Tm (Fig 1B and Table 1), indicating destabilization of CRMP-2 by a high concentration of divalent cations Test for heat stability of CRMP-2 using the Thermofluor method A series of conditions were screened in 96-well format, by measuring the fluorescence of SyproOrange, in order to further characterize the stability of CRMP-2 in the presence and absence of divalent cations The results clearly indicate that, although 20 mm CaCl2 and MgCl2 stabilize the protein slightly, a 200 mm concentration destabilizes the protein significantly (Fig 1D,E and Table 1) The same effect was observed in two different buffers, phosphate and Hepes, both adjusted to pH 7.5 The results are summarized in Table Divalent cations promote the tetramerization of CRMP-2 The oligomerization status of CRMP-2 was analysed by gel filtration in the presence and absence of 20 mm CaCl2 and MgCl2 It is evident that, in the absence of divalent cations, only approximately 50% of CRMP-2 is in the tetrameric state The rest of CRMP-2 is in its monomeric form In the presence of either Ca2+ or FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al Structural properties of CRMP-2 A B C Fig Folding and stability of CRMP-2 (A) CD spectra for CRMP-2 in different buffers The spectra were measured at 23.4 °C as described in the Experimental procedures The samples are: ⁄ 50 mM NaCl (thin ⁄ thick black line); 200 ⁄ 20 mM CaCl2 (thin ⁄ thick red line); and 200 ⁄ 20 mM MgCl2 (thin ⁄ thick blue line) in 10 mM Hepes (pH 7.3) (B) Melting curves based on the change in molar ellipticity as a function of temperature Sample colours are as described in (A) (C) Spectra for CRMP-2 after heating denaturation, measured at 90 °C The samples contain 200 mM CaCl2 (red), 200 mM MgCl2 (blue), or no additives (black) in 10 mM Hepes (pH 7.3) In the absence of divalent cations, CRMP-2 forms a b-aggregate (D) Thermofluor stability assay Eight replicate samples are shown at a single condition with 50 mM phosphate buffer + 20 mM CaCl2 The curves were normalized such that the maximum is and the minimum is (E) Superposition of averaged Thermofluor curves from samples under the conditions: phosphate buffer (thin black line); phosphate + 150 mM NaCl (thick black line); phosphate + 20 mM CaCl2 (thick red line); phosphate + 200 mM CaCl2 (thin red line); phosphate + 20 mM MgCl2 (thick blue line); and phosphate + 200 mM MgCl2 (thin blue line) D Mg2+, however, the protein is almost completely tetrameric (Fig 2A), with only a minor fraction of monomer being detectable The structure of CRMP-2 in the absence of calcium ions The replacement of calcium with magnesium in CRMP-2 crystallization gave rise to a novel orthorhombic crystal form, which diffracted X-rays signifi- E cantly better than the monoclinic form previously obtained in the presence of Ca2+ [5] Thus, the human CRMP-2 structure could be refined to a resolution of ˚ 1.9 A (Fig 2B and Table 2) As a slight drawback, a significant pseudotranslational symmetry component was present in the new crystal form (see below), which lead to unusually high R factors in refinement The anomaly of the crystal form is given rise to by the noncrystallographic symmetry axes present in the CRMP-2 tetramer FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4585 Structural properties of CRMP-2 V Majava et al Table Tm values from CD spectroscopy and Thermofluor assays In both cases, the Tm was taken as the point of steepest ascent of the measured curve The number of replicates for each condition in the Thermofluor assays is given in parentheses Assay CD (all in 10 mM Hepes, pH 7.3) 20 mM CaCl2 200 mM CaCl2 20 mM MgCl2 200 mM MgCl2 Thermofluor 50 mM Hepes (pH 7.5) 150 mM NaCl 20 mM CaCl2 200 mM CaCl2 20 mM MgCl2 200 mM MgCl2 50 mM NaPO4 (pH 7.5) 150 mM NaCl 20 mM CaCl2 200 mM CaCl2 20 mM MgCl2 200 mM MgCl2 Tm 49.5 44 48.5 45.5 46.1 47.6 47.3 40.6 47.5 44.9 46.8 47.5 47.5 39.8 47.5 40.3 ± ± ± ± ± ± ± ± ± ± ± ± 0.9 (8) 0.4 (7) 0.4 (6) 2.0 (6) (6) 0.5 (7) 0.8 (6) (8) (8) 0.8 (6) (6) 1.1 (7) As previously described [5], CRMP-2 forms a homotetramer by an arrangement with 222 symmetry (i.e a ‘dimer of dimers’) (Fig 3B) We have also previously suggested that this type of oligomeric assembly is responsible for dimer formation between homodimers of CRMP-1 and CRMP-2, resulting in a heterotetramer [5] The protein used in both the current and previous [5] structural studies contains a His-tag, which is disordered in the crystal structure; thus, it is highly unlikely that the oligomeric status or stability of CRMP-2 would be affected by the affinity tag Analysis of the packing Pseudotranslation with the vector 0,0.175,0.5 (fraction 26.3%) was clearly observed in the data obtained from the orthorhombic crystal form, indicating that a fraction of the tetramers in the unit cell are related to each other by this vector This was confirmed by the solved structure, in which, for each of the four tetramers in the unit cell, there is another one related by pseudotranslation Pseudotranslation affects refinement statistics by incorporating a large amount of weak reflections and, thus, leads to the crystallographic R factors during refinement being higher than generally expected for the used resolution range Taking this into account, the observed Rcryst and Rfree values from this crystal form are acceptable An analysis of the data using rstats software [31] also confirmed that the weak reflections in the data had systematically high 4586 A B Fig Divalent cations and the oligomeric structure of CRMP-2 (A) Analysis of oligomeric state by size exclusion chromatography The samples contained either no additives (black), 20 mM CaCl2 (red), or 20 mM MgCl2 (blue) The elution volumes of molecular mass markers (in kDa) are indicated above the graph (B) The crystal structure of CRMP-2 and the locations of detected divalent cations Ca2+ ions (from the previous structure) [5] are shown in red and Mg2+ (from the current structure) are shown in blue, and the different subunits of CRMP-2 are colour-coded Only the ions bound to the two monomers in front are visible in this view crystallographic R factors (data not shown) A better quality indicator in such a case is the correlation coefficient, and these values indicate that the CRMP-2 model is accurate (Table 2) Comparison with the structure in the presence of calcium The high-resolution structure obtained from the orthorhombic crystal form grown in the presence of Mg2+, but in the absence of Ca2+, was compared with the FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al Structural properties of CRMP-2 Table Crystallographic data collection and structure refinement Space group P212121 Unit cell parameters Data collection ˚ Resolution (A) Completeness (%) Rmerge (%) Redundancy Structure refinement Rcryst (%) Rfree (%) ˚ rmsd bond lengths (A) rmsd bond angles (°) Correlation coefficient Correlation coefficient (free) ˚ 86.5, 126.2, 209.8 A 20–1.90 (2.00–1.90) 94.2 (83.7) 12.1 (46.6) 11.4 (3.8) 6.0 (5.5) 25.6 32.5 0.016 1.6 0.915 0.847 previous structure determined in the presence of Ca2+ The Ca rmsd between the new structure and the pre˚ vious one is 0.2–0.35 A, depending on which chains of the tetramer are compared The rmsd of Ca positions ˚ for the whole tetramer is 0.32 A, as determined by ssm [32] The electron density clearly indicates that no large ion is bound to the previously observed calcium site [5] in the new crystal form This observation further confirms the presence of a Ca2+ ion in the previous structure because the main difference between the crystallization conditions is the change of Ca2+ to Mg2+ The solvent environment of the new crystal structure was analysed to identify putative Mg2+ ions The only location that shows an electron density reminiscent of hydrated Mg2+, as well as a suitable coordination environment, is approximately the same site where Ca2+ was bound in the earlier structure, at the mouth of the central pocket in the (ba)8 barrel (Fig 2B) It should be noted that Mg2+ is not easy to distinguish from a water molecule based on electron density alone because it has the same number of electrons as oxygen A C B Fig Small-angle X-ray scattering (A) Superposition of the measured scattering curves in the presence (red) and absence (green) of 20 mM CaCl2, as well as the theoretical scattering curve calculated from the crystal structure (black) using CRYSOL (B) Distance distribution functions for CRMP-2 in the presence (red) and absence (green) of CaCl2 (C) Ab initio models from DAMMIN (spheres) superimposed on the crystal structure (ribbons) Red spheres indicate the SAXS structure in the presence of Ca2+, and green spheres indicate the structure in its absence FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4587 Structural properties of CRMP-2 V Majava et al Considering the effects of Ca2+ on CRMP-2 oligomerization and stability, the solvent shell of the earlier CRMP-2 crystal structure (Protein Data Bank entry 2GSE) [5] was carefully re-analysed to locate any calcium ions, as indicated by an electron density too strong for a water molecule and a good coordination environment for calcium, that might explain such effects Indeed, two novel calcium sites were identified in each CRMP-2 monomer These are located between the backbone carbonyls of residues 128 and 464 and near the side chain of Gln245, which is located at an oligomerization interface (Fig 2B) In the structure determined in the presence of Mg2+, these sites not contain Mg2+ ions Small-angle X-ray scattering The oligomeric status and solution structure of CRMP-2 were studied using synchrotron SAXS in the presence and absence of Ca2+ In line with the above results, SAXS indicated a tetrameric structure for the sample measured in the presence of CaCl2 (Fig and Table 3) The estimated molecular mass of 191 kDa, based on a standard sample of BSA, also clearly shows the predominant presence of a tetrameric form (expected molecular mass of 220 kDa) The crystal structure could easily be fitted into ab initio models built based on the distance distribution function calculated from the SAXS data (Fig 3B,C) Similar results were also obtained in the absence of calcium, indicating that the major form of CRMP-2 is a tetramer also in the absence of Ca2+, in the high protein concentration conditions employed (10 mgỈmL)1) However, when calculating a theoretical scattering curve based on the crystal structure, the fit between the measured data and the crystal Table SAXS results The calculated values were obtained from samples of 10 mgỈmL)1, and the crystal structure values for Rg, excluded volume and Dmax are as given by CRYSOL The theoretical I(0) was calculated for the crystal structure based on the I(0) of BSA The SAXS excluded volumes are the average volumes of the ab initio models obtained from DAMMIN Sample Rg (nm) I(0) Excluded Molecular Dmax volume mass (nm) (nm3) (kDa) CRMP-2 + 3.65 ± 0.001 716 ± 0.4 10.5 310 CaCl2 CRMP-2 3.75 ± 0.004 708 ± 0.4 11.5 284 Crystal 3.69 825 12.5 261 structure (homotetramer) 4588 191 189 220 structure is better in the presence of calcium (Fig 3A), indicating a possible heterogeneity in the sample without calcium Indeed, the software oligomer could fit the data without calcium better when given a combination of calculated scattering curves for a tetramer and a dimer or a monomer This was not true for the sample containing CaCl2 (data not shown) In line with this, ab initio model building with dammin resulted in a better fit to the crystal structure for the sample in the presence of CaCl2, indicating the possibility of subtle calcium-dependent movements of the subunits, or the presence of dimeric or monomeric forms of CRMP-2 in the sample without calcium These observations could also explain the success of crystallization experiments only in the presence of Ca2+ or Mg2+ Analysis of ion binding by surface plasmon resonance To obtain an idea of the extent and affinity of divalent cation binding by CRMP-2, we carried out a surface plasmon resonance experiment where CRMP2 was immobilized and incubated in the presence of different concentrations of MgCl2, CaCl2, BaCl2 and KCl (Fig 4) The results indicate that the divalent cations produce a similar strong response, whereas potassium chloride only gives a weak signal The observed strong signal suggests the presence of many binding sites, the binding involving hydrated ions and ⁄ or ordering of solvent on the protein surface The estimated overall Kd values for the binding of the cations onto the CRMP-2 protein surface are 15–20 mm for the divalent cations and > 100 mm for potassium The detection of several isoforms of CRMP-2 in rat hippocampus using 2D electrophoresis and MS When identifying all the protein spots from rat hippocampus on 2D gels [30], 26 spots were identified as CRMP-2 (Fig 5) To shed light on the post-translational modifications present in CRMP-2, all these spots were picked, digested with trypsin and analysed by nano-LC-ESI-MS ⁄ MS The results are summarized in the Supporting information (Table S1) It is noteworthy that the deamidation of Asn356 was detected in eight spots, and that oxidation of methionines 64, 152, 168, 362, 375 and 437 was detected in at least five spots each We also detected phosphorylation of Thr509 in three spots (one of these spots also showed phosphorylation of Ser522) FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al Structural properties of CRMP-2 A B Fig 2D gel electrophoretic analysis of rat brain hippocampal proteins; spots identified as CRMP-2 are highlighted The details on the selected spots are given in Table S1 In the inset, the region containing the CRMP-2 spots is shown in more detail Fig Surface plasmon resonance analysis of ion binding by CRMP-2 (A) Kinetic analysis of Ca2+ binding by immobilized CRMP-2 For clarity, only injections with 1, 5, 10 and 50 mM CaCl2 are shown (B) A comparison of the responses obtained with different ions at 20 mM concentration Ca2+, red; Mg2+, blue; Ba2+, light blue; K+, orange Mapping of the post-translational modifications onto the crystal structure of CRMP-2 A number of different post-translational modifications were identified for the various isoforms detected for adult rat brain hippocampal CRMP-2 (see above) These modifications were mapped onto the 3D structure of human CRMP-2 (Fig 6) The sequence identity between human and rat CRMP-2 is very high, with only seven residues out of 572 being nonconserved, and all the detected modification sites being conserved All the post-translational modifications are located outside the core of the (ba)8 barrel fold, mainly in looplike structures on the protein surface The sites are concentrated on specific regions in 3D space, especially Fig Mapping of post-translational modifications onto the 3D structure of CRMP-2 In this stereo view, the major oxidation sites are shown in magenta, minor oxidation sites are shown in yellow, major deamidation sites are shown in green, and minor deamidation sites are shown in blue when considering the sites that were detected in several spots of the 2D gel (see Discussion) Discussion The effects of divalent cations on the stability and oligomeric status of CRMP-2 CRMP-2 forms homotetramers [5,11] and, in the present study, these assemblies are demonstrated to be stabilized FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4589 Structural properties of CRMP-2 V Majava et al by divalent cations The heat stability of CRMP-2 was shown, by two independent methods, to decrease significantly when the divalent cation concentration was raised from 20 to 200 mm This indicates that the effect on CRMP-2 stability is dual: at low concentration, both Ca2+ and Mg2+ stabilize the protein, whereas, at higher concentrations, the effect is the opposite Divalent cations have previously been shown to similarly affect protein stability in a concentrationdependent manner [33–40] For example, reported examples include glucose oxidase [35], RNase T1 [36] and papain [34], and the results can often be explained by two different modes of binding of divalent cations to proteins At lower concentrations, the hydrated ions act mainly to order and stabilize the solvent shell around the protein molecule At a higher concentration, the ions will bind directly to the protein surface, resulting in destabilization The structure of CRMP-2 The crystal structure of human CRMP-2 was refined at ˚ 1.9 A resolution, and the overall structure is very similar to the earlier lower resolution structure obtained in the presence of calcium [5], also demonstrating the same oligomeric assembly into a homotetramer We have also shown by SAXS studies that the homotetrameric structure of CRMP-2 is maintained in solution, especially in the presence of calcium The good fit between our experimental solution and crystal structures indicates that the crystal structure is an accurate representation of the CRMP-2 tetramer in solution Moreover, the conformation obtained by ab initio modelling resembles the crystal structure more closely in the presence of calcium than in its absence This hints at the possibility of subtle conformational changes within the tetramer that can be induced by divalent cations In principle, in the presence of calcium, the CRMP-2 tetramer appears to be slightly more compact than in its absence, as demonstrated by the smaller Rg and Dmax values for the Ca2+-containing sample in conjunction with the I(0) and excluded volumes, which are higher (Table 3) These differences could also be caused, at least partly, by the presence of small amounts of dimeric CRMP-2 in the absence of calcium Using CD spectroscopy, we have shown that CRMP-2 undergoes a transition to a b-aggregate upon heating in the absence of divalent cations Interestingly, CRMP-2 has been characterized as a constituent of the paired helical filaments of neurofibrillary tangles in Alzheimer’s disease [41,42] Future studies should aim to investigate any possible relationships between the folding and denaturation behaviour of CRMP-2 4590 and neuronal fibril formation in Alzheimer’s disease brains Post-translational modifications and previously characterized mutations of CRMP-2 A number of post-translational modifications were detected in CRMP-2 from rat brain, including several sites of deamidation, oxidation and phosphorylation (Table S1) Although both of the phosphorylation sites that were detected (i.e Thr509 and Ser522) were in the C-terminal tail region, which is predicted to be structurally disordered, most of the other modifications could be mapped onto the 3D structure Thr509 and Ser522 have been both characterized as major phosphorylation sites in CRMP-2 [9,42] The reason why some of the other previously characterized phosphorylation sites on the C-terminal tail were not detected could be due to the fact that hippocampal tissue from an adult animal was used This aspect, however, was not investigated further in the present study because we were specifically interested in the post-translational modifications that could be mapped onto the folded domain of CRMP-2 Previously, modifications of residues within the folded region have not been characterized in detail, except for the detection of one phosphorylation site at Ser465 [43] Three ‘hotspot’ regions for post-translational modifications can be visualized in the 3D structure of CRMP2 (Fig 6) The first concerns the main deamidation site and four of the main oxidation sites The second one contains the remaining two main oxidation sites and two minor deamidation sites, and the third one a minor deamidation site and three minor oxidation sites The reasons for the concentration of the modifications to these areas in 3D space are currently not known, but they could include the accessibility of these areas towards modifications, their mobility and the relation of such modifications towards CRMP-2 function In light of the finding that CRMP-2 is one of the neuronal proteins that accumulate high levels of isoaspartate [23], it is interesting to observe a number of deamidated asparagine residues in CRMP-2 The formation of isoaspartate is an important source of protein damage under physiological conditions, and is linked to the deamidation of Asn residues The deamidation of Asn356, detected in eight of the 26 characterized CRMP-2 spots, could be related to isoaspartate accumulation on brain CRMP-2 The e204 mutation in the Caenorhabditis elegans unc-33 gene [44] results in a protein where a conserved aspartate residue is mutated into an asparagine; the corresponding residue in CRMP-2 is Asp71 [44] In a FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al Structural properties of CRMP-2 yeast two-hybrid assay, the mutation prevented unc-33 oligomerization [45] The mutant was originally isolated by identifying its involvement in paralysis resulting from defective axon growth [44], and similar effects on axonal growth have subsequently been found with the corresponding D71N mutant of CRMP-2 [7] In the 3D structure, Asp71 is buried within the folded protein, close to the interface between the small and large lobes of CRMP-2, but far from the tetramerization interfaces Its side chain is buried, being close to that of Arg361, but no hydrogen bond ⁄ salt bridge is formed between the two side chains These two buried charges apparently neutralize each other and, in the D71N mutant, the charged Arg361 is expected to have an isolated buried charge, which may destabilize the protein buffer, which was also used for the measurement of a buffer control by CD CD spectra were measured in the wavelength range 195–250 nm, on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) The protein concentration was lm, and a mm cuvette was used After measuring each spectrum at 23.4 °C, a temperature scan at a fixed wavelength of 220 nm was run between 30 and 70 °C to obtain a melting curve After the temperature scan, CD spectra were further recorded for the samples, both at 90 °C and after cooling back to room temperature The effects of the following additives on the behaviour of CRMP-2 were studied: 20 or 200 mm CaCl2, 20 or 200 mm MgCl2, and 50 mm NaCl CD spectra and a temperature scan were recorded using exactly the same parameters and procedures for all samples Of note, the sample gained a gel-like appearance upon heating in the absence of divalent cations, but not in their presence Conclusions Stability analysis by the Thermofluor method CRMP-2 is a central regulator of axonal growth cone guidance, and its function is likely to involve both homo- and heterotetramerization, as well as post-translational modifications Using a number of biochemical and biophysical methods, we have gained a more detailed view than was previously available for the structure and properties of the CRMP-2 protein, both in solution and in the crystal state Divalent cations have a drastic effect on both the stability and oligomeric state of CRMP-2, and a number of different post-translationally modified isoforms of CRMP-2 could be identified in the brain Our data, including the mapping of post-translational modifications onto the structure of CRMP-2, open up new possibilities for studying the function and interactions of CRMP-2, which still remain enigmatic The thermal stability of CRMP-2 was analysed in 96-well format by following the fluorescence from SYPRO Orange (Invitrogen, Carlsbad, CA, USA) as a function of temperature (i.e the so-called Thermofluor or thermal shift assay method) [46,47] The experiment was carried out using a 7500 Real Time PCR System apparatus (Applied Biosystems, Foster City, CA, USA) and the temperature was scanned from 20 to 90 °C with °C increments, with monitoring of fluorescence at 542 nm Eight replicates each from 12 different conditions were randomly placed on the 96-well PCR plate Occasionally, curves with abnormal shapes were observed; such curves were excluded from the analysis, most likely resulting from incomplete sealing of an individual well on the 96-well PCR plate Experimental procedures Protein purification The purification of human CRMP-2 has been described previously [5] The protein batches for the present study were obtained from the SGC Stockholm laboratory A construct containing residues 1–490 was used for the surface plasmon resonance experiments, and other experiments were carried out with a construct containing residues 13–490 of CRMP-2 The His tags were not removed prior to any experiments CD spectroscopy CRMP-2 was extensively dialysed against 10 mm Hepes (pH 7.3); subsequent dilutions were made in the dialysis Size exclusion chromatography The oligomeric status of CRMP-2 was analysed by gel filtration on a Superdex 200 HR 10 ⁄ 30 column coupled to an ă AKTApurier (GE Healthcare, Uppsala, Sweden) An identical sample (0.67 mgỈmL)1 of 200 lL) was run with the same protocol in 10 mm Hepes (pH 7.5), 100 mm NaCl, and in the same buffer containing either 20 mm CaCl2 or 20 mm MgCl2 Sample elution was followed at 280 nm Molecular masses were estimated by comparing the sample elution volumes with those observed for standard proteins run on the same column Crystallization, data collection and structure solution Human CRMP-2 was crystallized essentially as described previously [5], while simultaneously screening for substitutes for CaCl2 that could promote crystallization In the absence FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4591 Structural properties of CRMP-2 V Majava et al of CaCl2, the only tested additive producing crystals was MgCl2 Crystals (crystal form 1) without CaCl2 were grown at 20 °C in hanging drops over a well solution consisting of 15% poly(ethylene glycol) 10 000, 0.1 m Tris (pH 9), 0.2 m MgCl2 and 10 mm MgI2 Another crystal form (crystal form 2) was similarly grown in the absence of MgI2 (pH 8.5) Prior to data collection, the crystals were briefly soaked in a cryoprotectant solution (well solution supplemented with 20% glycerol) and cooled to 100 K in a stream of gaseous nitrogen Diffraction data were collected at MAXLab (Lund, Sweden) beamline I911-2 [48] Data processing was performed using xds [49] and xdsi [50] An analysis of the data revealed the presence of pseudotranslational symmetry in crystal form (26.3%, fractional translation 0,0.175,0.5) However, space group determination for crystal form was not straightforward, and this monoclinic crystal was found to be pseudomerohedrally twinned; for this reason, crystal form was discarded from the analysis The previously determined structure of human CRMP-2 [5] was used as a template in molecular replacement Four monomers of CRMP-2 were found within the asymmetric unit using molrep [51], and refinement and model building were carried out iteratively using refmac [52], phenix refine [53,54] and coot [55] The coordinates and structure factors were deposited in the Protein Data Bank under the accession code 2VM8 Surface plasmon resonance CaCl2, at concentrations in the range 1–10 mgỈmL)1, were measured on the EMBL Hamburg ⁄ DESY beamline X33, and the corresponding buffer was always used for a blank experiment Programs from the atsas software package [56] were used for data analysis, essentially as described previously [57] The measured data were further processed using primus [58] The molecular mass was estimated by comparing the forward scattering I(0) with that of a standard solution of BSA The distance distributions were obtained using gnom [59] and further used for ab initio modelling in dammin [60] An averaged model was generated from several runs using damaver [61], and the SAXS model and the crystal structure were superimposed with supcomb [62] The possible oligomeric assemblies were also studied using oligomer [58], after evaluating the solution scattering of each possible component using crysol [63] 2D electrophoresis and MS Materials Immobilized pH gradient strips and buffers were purchased from Amersham Biosciences, a part of GE Healthcare (Milwaukee, WI, USA) Reagents for polyacrylamide gel preparation were purchased from Bio-Rad Laboratories (Hercules, CA, USA) Chaps was obtained from Roche Diagnostics (Mannheim, Germany), urea was obtained from AppliChem (Darmstadt, Germany), thiourea was obtained from Fluka (Buchs, Switzerland), 1,4-dithioerythritol and EDTA were obtained from Merck (Darmstadt, Germany) and tributylphosphine was obtained from Pierce Biotechnology (Rockford, IL, USA) The binding of Ca2+, Mg2+, Ba2+ and K+ by CRMP-2 was analysed by surface plasmon resonance on a Biacore 3000 apparatus (Biacore AB, Uppsala, Sweden) by immobilizing CRMP-2 on a CM5 chip and passing 0, 1, 5, 10, 20, 50 and 100 mm solutions of CaCl2, MgCl2, BaCl2 and KCl over the chip In the immobilization, 10 mm socium acetate buffer (pH 4.5) was used During the binding experiment, the running buffer contained 10 mm Hepes (pH 7.5), 100 mm NaCl and 0.004% surfactant P20 (Biacore AB), in addition to the salts being tested The experiments were carried out at 25 °C, with a flow rate of 30 lLỈmin)1 For regeneration of the surface between injections, the ions were allowed to dissociate freely into the binding buffer A control channel on the chip was similarly treated, with the exception that no protein was immobilized onto it The data were fitted against a : binding model using biaevaluation software (Biacore AB) Twenty-three-month-old rat hippocampus tissue was powderized in liquid nitrogen and suspended in mL of sample buffer [20 mm Tris, m urea, m thiourea, 4% Chaps, 10 mm 1,4-dithioerythritol, mm EDTA and mm phenylmethanesulfonyl fluoride, containing tablet CompleteÔ (Roche Diagnostics) and 0.2% (v ⁄ v) phosphatase inhibitor cocktail (Calbiochem, San Diego, CA, USA)] The suspension was sonicated for approximately 30 s and centrifuged at 15 000 g for 60 at 12 °C Desalting was performed with an Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA, USA), with a molecular mass cut-off of 10 kDa The protein concentration of the supernatant was determined by the Bradford assay Small-angle X-ray scattering 2D gel electrophoresis For SAXS, CRMP-2 was dialysed into a buffer containing 10 mm Hepes (pH 7.5) and 100 mm NaCl SAXS data for CRMP-2 in the presence and absence of 20 mm Samples of 750 lg of protein were applied on immobilized nonlinear pH gradient (pH 3–10) strips Focusing started at 4592 Sample preparation FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al Structural properties of CRMP-2 200 V, and the voltage was gradually increased to 8000 V at VỈmin)1 and kept constant for a further h (approximately 150 000 Vh in total) Separation in the second dimension was performed on a 10–16% gradient SDS ⁄ PAGE After protein fixation for 12 h in 50% methanol and 10% acetic acid, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for h, and the excess of dye was removed with distilled water Molecular masses were determined by running standard protein markers (Bio-Rad Laboratories), covering the range 10–250 kDa pI values were used as provided by the supplier of the immobilized pH gradient strips against the SwissProt 51.0 database to identify the protein spot The search parameters were set: a mass tolerance of 500 p.p.m for MS tolerance, 0.2 Da for MS ⁄ MS tolerance, one missing cleavage site, fixed modification of carbamidomethyl, and variable modification of methionine oxidation Positive protein identifications were based on a significant Mowse score [64] After the protein was identified, an error-tolerant search was performed to detect unspecific cleavage and unassigned modifications Protein identification and modification returned from mascot were manually examined and filtered to create a confirmed protein identification and modification list In-gel digestion Acknowledgements Spots of interest were excised and washed sequentially with 10 mm ammonium bicarbonate and 50% acetonitrile in 10 mm ammonium bicarbonate After washing, the gel plugs were shrunk by the addition of acetonitrile and dried in a SpeedVac (Eppendorf, Hamburg, Germany) The dried gel pieces were reswollen with 40 ngỈlL)1 trypsin (sequencing grade; Promega, Madison, WI, USA) in digestion buffer (5 mm octyl b-d-glucopyranoside and 10 mm ammonium bicarbonate) and incubated for h at 30 °C Extraction was performed first with 10 lL of 1% trifluoroacetic acid in mm octyl b-d-glucopyranoside, and then using 10 lL of 0.1% trifluoroacetic acid, 4% acetonitrile Both peptide extracts were pooled and concentrated in a SpeedVac until the volume reached lL P K is an Academy Research Fellow (Academy of Finland) The work was supported by grants from the Finnish MS Foundation and the Department of Biochemistry, Oulu University (V M.) The support of Ylva Lindqvist and Gunter Schneider in the early stages of this work and enlightening discussions with Inari Kursula and Maxim Petoukhov are gratefully acknowledged We thank the Stockholm node of the SGC for providing materials for this study and the beamline staff at EMBL Hamburg and MAX-Lab for enabling smooth data collection The measurement of synchrotron SAXS data at EMBL Hamburg and the crystallographic data collection at MAX-Lab were both supported by the European Community – Research Infrastructure Action under the FP6 ‘Structuring the European Research Area’ Programme (through the Integrated Infrastructure Initiative ‘Integrating Activity on Synchrotron and Free Electron Laser Science’), contract RII3-CT-2004-506008 (IA-SFS) Protein identification and characterization by nano-LC-ESI-MS ⁄ MS Six microlitres of the extracted sample were used for nanoLC-ESI-MS ⁄ MS investigation The HPLC used comprised an Ultimate 3000 system (Dionex Corporation; Sunnyvale, CA, USA) equipped with a PepMap C-18 analytic column (75 lm · 150 mm) The gradient was (A = 0.1% formic acid in water, B = 80% acetonitrile ⁄ 0.08% formic acid in water) 4% B to 60% B from to 30 min, 90% B from 30 to 35 min, and 4% B from 35 to 60 Peptide spectra were recorded over the mass range of m ⁄ z 350–1600, and MS ⁄ MS spectra were recorded in an information dependent data acquisition over the mass range of m ⁄ z 50–1600 Repeatedly, one MS spectrum was recorded followed by two MS ⁄ MS spectra on the QSTAR XL instrument (Applied Biosystems); the accumulation time was s for MS spectra and s for MS ⁄ MS spectra The collision energy was automatically set, according to the mass and charge state of the peptides chosen for fragmentation Doubly or triply charged peptides were chosen for MS ⁄ MS experiments due to their good fragmentation characteristics MS ⁄ MS spectra were interpreted using mascot software (Matrix Science Ltd, London, UK) and searched References Kamata T, Subleski M, Hara Y, Yuhki N, Kung H, Copeland NG, Jenkins NA, Yoshimura T, Modi W & Copeland TD (1998) Isolation and characterization of a bovine neural specific protein (CRMP-2) cDNA homologous to unc-33, a C elegans gene implicated in axonal outgrowth and guidance Brain Res Mol Brain Res 54, 219–236 Schmidt EF & Strittmatter SM (2007) The CRMP family of proteins and their role in Sema3A signaling Adv Exp Med Biol 600, 1–11 Yoshimura T, Arimura N & Kaibuchi K (2006) Molecular mechanisms of axon specification and neuronal disorders Ann NY Acad Sci 1086, 116–125 Charrier E, Reibel S, Rogemond V, Aguera M, Thomasset N & Honnorat J (2003) Collapsin response mediator proteins (CRMPs): involvement in nervous system FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4593 Structural properties of CRMP-2 10 11 12 13 14 15 V Majava et al development and adult neurodegenerative disorders Mol Neurobiol 28, 51–64 Stenmark P, Ogg D, Flodin S, Flores A, Kotenyova T, Nyman T, Nordlund P & Kursula P (2007) The structure of human collapsin response mediator protein 2, a regulator of axonal growth J Neurochem 101, 906–917 Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimura T, Shiromizu T, Watanabe H, Inagaki N, Iwamatsu A, Hotani H et al (2002) CRMP-2 binds to tubulin heterodimers to promote microtubule assembly Nat Cell Biol 4, 583–591 Kawano Y, Yoshimura T, Tsuboi D, Kawabata S, Kaneko-Kawano T, Shirataki H, Takenawa T & Kaibuchi K (2005) CRMP-2 is involved in kinesin-1dependent transport of the Sra-1 ⁄ WAVE1 complex and axon formation Mol Cell Biol 25, 9920–9935 Nishimura T, Fukata Y, Kato K, Yamaguchi T, Matsuura Y, Kamiguchi H & Kaibuchi K (2003) CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth Nat Cell Biol 5, 819–826 Brown M, Jacobs T, Eickholt B, Ferrari G, Teo M, Monfries C, Qi RZ, Leung T, Lim L & Hall C (2004) Alpha2-chimaerin, cyclin-dependent kinase ⁄ p35, and its target collapsin response mediator protein-2 are essential components in semaphorin 3A-induced growth-cone collapse J Neurosci 24, 8994–9004 Lee S, Kim JH, Lee CS, Kim JH, Kim Y, Heo K, Ihara Y, Goshima Y, Suh PG & Ryu SH (2002) Collapsin response mediator protein-2 inhibits neuronal phospholipase D(2) activity by direct interaction J Biol Chem 277, 6542–6549 Wang LH & Strittmatter SM (1997) Brain CRMP forms heterotetramers similar to liver dihydropyrimidinase J Neurochem 69, 2261–2269 Beyreuther BK, Freitag J, Heers C, Krebsfanger N, Scharfenecker U & Stohr T (2007) Lacosamide: a review of preclinical properties CNS Drug Rev 13, 21–42 Bisgaard CF, Jayatissa MN, Enghild JJ, Sanchez C, Artemychyn R & Wiborg O (2007) Proteomic investigation of the ventral rat hippocampus links DRP-2 to escitalopram treatment resistance and SNAP to stress resilience in the chronic mild stress model of depression J Mol Neurosci 32, 132–144 Nakata K, Ujike H, Sakai A, Takaki M, Imamura T, Tanaka Y & Kuroda S (2003) The human dihydropyrimidinase-related protein gene on chromosome 8p21 is associated with paranoid-type schizophrenia Biol Psychiatry 53, 571–576 Mimura F, Yamagishi S, Arimura N, Fujitani M, Kubo T, Kaibuchi K & Yamashita T (2006) Myelinassociated glycoprotein inhibits microtubule assembly by a Rho-kinase-dependent mechanism J Biol Chem 281, 15970–15979 4594 16 Tahimic CG, Tomimatsu N, Nishigaki R, Fukuhara A, Toda T, Kaibuchi K, Shiota G, Oshimura M & Kurimasa A (2006) Evidence for a role of collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells Biochem Biophys Res Commun 340, 1244–1250 17 Wu CC, Chen HC, Chen SJ, Liu HP, Hsieh YY, Yu CJ, Tang R, Hsieh LL, Yu JS & Chang YS (2008) Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes Proteomics 8, 316–332 18 Kimura T, Watanabe H, Iwamatsu A & Kaibuchi K (2005) Tubulin and CRMP-2 complex is transported via kinesin-1 J Neurochem 93, 1371–1382 19 Kanninen K, Goldsteins G, Auriola S, Alafuzoff I & Koistinaho J (2004) Glycosylation changes in Alzheimer’s disease as revealed by a proteomic approach Neurosci Lett 367, 235–240 20 Petratos S, Li QX, George AJ, Hou X, Kerr ML, Unabia SE, Hatzinisiriou I, Maksel D, Aguilar MI & Small DH (2008) The b-amyloid protein of Alzheimer’s disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP mechanism Brain 131, 90–108 21 Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, Ihara Y, Mikoshiba K et al (2005) Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer’s disease Genes Cells 10, 165–179 22 Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A & Kaibuchi K (2005) GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity Cell 120, 137–149 23 Zhu JX, Doyle HA, Mamula MJ & Aswad DW (2006) Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain J Biol Chem 281, 33802–33813 24 Arimura N, Inagaki N, Chihara K, Menager C, Nakamura N, Amano M, Iwamatsu A, Goshima Y & Kaibuchi K (2000) Phosphorylation of collapsin response mediator protein-2 by Rho-kinase Evidence for two separate signaling pathways for growth cone collapse J Biol Chem 275, 23973–23980 25 Arimura N, Menager C, Kawano Y, Yoshimura T, Kawabata S, Hattori A, Fukata Y, Amano M, Goshima Y, Inagaki M et al (2005) Phosphorylation by Rho kinase regulates CRMP-2 activity in growth cones Mol Cell Biol 25, 9973–9984 26 Castegna A, Aksenov M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR & Butterfield DA (2002) Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain Part II: FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS V Majava et al 27 28 29 30 31 32 33 34 35 36 37 38 dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71 J Neurochem 82, 1524–1532 Choi J, Forster MJ, McDonald SR, Weintraub ST, Carroll CA & Gracy RW (2004) Proteomic identification of specific oxidized proteins in ApoE-knockout mice: relevance to Alzheimer’s disease Free Radic Biol Med 36, 1155–1162 Pamplona R, Dalfo E, Ayala V, Bellmunt MJ, Prat J, Ferrer I & Portero-Otin M (2005) Proteins in human brain cortex are modified by oxidation, glycoxidation, and lipoxidation Effects of Alzheimer disease and identification of lipoxidation targets J Biol Chem 280, 21522–21530 Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce WM, Klein JB, Merchant M, Markesbery WR & Butterfield DA (2006) Redox proteomics identification of oxidized proteins in Alzheimer’s disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD Neurobiol Aging 27, 1564–1576 Chen WQ, Kang SU & Lubec G (2006) Protein profiling by the combination of two independent mass spectrometry techniques Nat Protoc 1, 1446–1452 Collaborative Computational Project, Number (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D Biol Crystallogr 50, 760–763 Krissinel E & Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr D Biol Crystallogr 60, 2256–2268 Xu X, Chen J, Zhang L, Wang S, Shen D & Liu Q (2007) Calcium ion-induced stabilization and refolding of agkisacutacin from Agkistrodon acutus venom studied by fluorescent spectroscopy J Fluoresc 17, 215– 221 Kaul P, Sathish HA & Prakash V (2002) Effect of metal ions on structure and activity of papain from Carica papaya Nahrung 46, 2–6 Akhtar MS, Ahmad A & Bhakuni V (2002) Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer Biochemistry 41, 7142–7149 Deswarte J, De Vos S, Langhorst U, Steyaert J & Loris R (2001) The contribution of metal ions to the conformational stability of ribonuclease T1: crystal versus solution Eur J Biochem 268, 3993–4000 Gotwals PJ, Chi-Rosso G, Ryan ST, Sizing I, Zafari M, Benjamin C, Singh J, Venyaminov SY, Pepinsky RB & Koteliansky V (1999) Divalent cations stabilize the alpha beta integrin I domain Biochemistry 38, 8280–8288 Ebel C, Faou P, Kernel B & Zaccai G (1999) Relative role of anions and cations in the stabilization of Structural properties of CRMP-2 39 40 41 42 43 44 45 46 47 48 49 50 51 52 halophilic malate dehydrogenase Biochemistry 38, 9039–9047 Rozhkov SP (1997) Stabilization of albumin by CaCl2- and MgCl2-regulated interaction of macromolecules: a study by the spin label method Biofizika 42, 1020–1028 Coolbear T, Whittaker JM & Daniel RM (1992) The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1 Biochem J 287, 367–374 Yoshida H, Watanabe A & Ihara Y (1998) Collapsin response mediator protein-2 is associated with neurofibrillary tangles in Alzheimer’s disease J Biol Chem 273, 9761–9768 Gu Y, Hamajima N & Ihara Y (2000) Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser518, and Ser-522 Biochemistry 39, 4267–4275 Vosseller K, Hansen KC, Chalkley RJ, Trinidad JC, Wells L, Hart GW & Burlingame AL (2005) Quantitative analysis of both protein expression and serine ⁄ threonine post-translational modifications through stable isotope labeling with dithiothreitol Proteomics 5, 388–398 Brenner S (1974) The genetics of Caenorhabditis elegans Genetics 77, 71–94 Tsuboi D, Hikita T, Qadota H, Amano M & Kaibuchi K (2005) Regulatory machinery of UNC-33 Ce-CRMP localization in neurites during neuronal development in Caenorhabditis elegans J Neurochem 95, 1629–1641 Cummings MD, Farnum MA & Nelen MI (2006) Universal screening methods and applications of ThermoFluor J Biomol Screen 11, 854–863 Ericsson UB, Hallberg BM, Detitta GT, Dekker N & Nordlund P (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies Anal Biochem 357, 289–298 Mammen CB, Ursby T, Thunnissen M & Als-Nielsen J (2004) Bent diamond crystals and multilayer based optics at the new 5-station protein crystallography beamline ‘Cassiopeia’ at MAX-lab AIP Conf Proc 705, 808–811 Kabsch W & Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogenbonded and geometrical features Biopolymers 22, 2577– 2637 Kursula P (2004) XDSi – a graphical interface for the data processing program XDS J Appl Crystallogr 37, 347–348 Vagin A & Teplyakov A (1997) MOLREP: an automated program for molecular replacement J Appl Crystallogr 30, 1022–1025 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS 4595 Structural properties of CRMP-2 53 54 55 56 57 58 59 V Majava et al maximum-likelihood method Acta Crystallogr D Biol Crystallogr 53, 240–255 Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK & Terwilliger TC (2002) PHENIX: building new software for automated crystallographic structure determination Acta Crystallogr D Biol Crystallogr 58, 1948–1954 Afonine PV, Grosse-Kunstleve RW & Adams PD (2005) A robust bulk-solvent correction and anisotropic scaling procedure Acta Crystallogr D Biol Crystallogr 61, 850–855 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics Acta Crystallogr D Biol Crystallogr 60, 2126–2132 Konarev PV, Petoukhov MV, Volkov VV & Svergun DI (2006) ATSAS 2.1, a program package for smallangle scattering data analysis J Appl Crystallogr 39, 277–286 Majava V, Petoukhov M, Hayashi N, Pirila P, Svergun ă D & Kursula P (2008) Interaction between the C-terminal region of human myelin basic protein and calmodulin: analysis of complex formation and solution structure BMC Struct Biol 8, 10 Konarev P, Volkov V, Sokolova A, Koch M & Svergun D (2003) PRIMUS – a Windows-PC based system for small-angle scattering data analysis J Appl Crystallogr 36, 1277–1282 Svergun D (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria J Appl Crystallogr 25, 495–503 4596 60 Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing Biophys J 76, 2879–2886 61 Volkov V & Svergun D (2003) Uniqueness of ab initio shape determination in small angle scattering J Appl Crystallogr 36, 860–864 62 Kozin M & Svergun D (2001) Automated matching of high- and low-resolution structural models J Appl Crystallogr 34, 33–41 63 Svergun D, Barberato C & Koch M (1995) CRYSOL – a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates J Appl Crystallogr 28, 768–773 64 Pappin DJ, Hojrup P & Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting Curr Biol 3, 327–332 Supporting information The following supplementary material is available: Table S1 Identification and characterization of rat CRMP2 (DPYL2_rat) by MS ⁄ MS spectrum This supplementary material can be found in the online version of this article Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary material supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 4583–4596 ª 2008 The Authors Journal compilation ª 2008 FEBS ... 7.3) 20 mM CaCl2 20 0 mM CaCl2 20 mM MgCl2 20 0 mM MgCl2 Thermofluor 50 mM Hepes (pH 7.5) 150 mM NaCl 20 mM CaCl2 20 0 mM CaCl2 20 mM MgCl2 20 0 mM MgCl2 50 mM NaPO4 (pH 7.5) 150 mM NaCl 20 mM CaCl2 20 0... the stability and oligomeric state of CRMP -2, and a number of different post-translationally modified isoforms of CRMP -2 could be identified in the brain Our data, including the mapping of post-translational. .. phosphate + 20 mM CaCl2 (thick red line); phosphate + 20 0 mM CaCl2 (thin red line); phosphate + 20 mM MgCl2 (thick blue line); and phosphate + 20 0 mM MgCl2 (thin blue line) D Mg2+, however, the protein