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Collapsin response mediator protein-5 (CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers in vivo and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca2+ and Mg2+, destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs.
The collapsin response mediator protein (CRMP) phosphoprotein family was first described as an intracellular component of the transduction pathway of the extracellular semaphorin 3A signal (Goshima et al. 1995). Its most recent member was simultaneously identified by three independent studies and reported under the names CRMP-5 (Fukada et al. 2000), CRAM (CRMP3-associated molecule) (Inatome et al. 2000), and unc33-like phosphoprotein 6 (Horiuchi et al. 2000). It is also sometimes referred to as dihydropyrimidinase-related protein 5. While the amino acid sequences of CRMPs 1-4 are ~70-75% identical, CRMP-5 shares ~50% sequence identity with CRMPs 1-4 and dihydropyrimidinase (DHPase) (Figure S1). It is thus equally distant from both non-enzymatic and enzymatic homologs and was suggested to be categorized into a third subfamily (Fukada et al. 2000). Within the protein family, several CRMPs (1, 2, and 4) occur in two alternatively spliced isoforms, with the less ubiquitously expressed ones containing an extra N–terminal domain of unknown structure and function.
CRMP-5 is crucial for and has unique roles in neuronal development (Veyrac et al. 2011; Yamashita et al. 2011; Camdessanché et al. 2012). Like all the CRMP family members, it is highly expressed in the developing brain as well as regions of neurogenesis of the adult brain (Fukada et al. 2000; Charrier et al. 2003). CRMP-5 itself has been identified as a biomarker in neuroendocrine lung cancer (Meyronet et al. 2008), while CRMP-5 autoantibodies (also named CV2) are known markers for paraneoplastic neurological syndromes and associated cancers, for example, small-cell lung cancer and thymoma (Yu et al. 2001; Rogemond and Honnorat 2000; Werry et al. 2009). Furthermore, CRMP-5 was shown to be induced as a result of neurotoxicity (Berg et al. 2011) supporting its proposed role in neurogenesis. The expression pattern of the CRMP-5 gene overlaps with that of one or several CRMPs in different cells, compartments, and stages of the neuronal development (Ricard et al. 2001; Charrier et al. 2003; Veyrac et al. 2005). It is thus hypothesized that CRMP-5 forms hetero-oligomers with and modulates the function of other CRMP family members (Fukada et al. 2000).
As an intracellular component of the semaphorin 3A signal transduction pathway, CRMP-5 is involved in regulating filopodial dynamics and growth cone development by negatively regulating the sensitivity of the growth cone to semaphorin 3A, (Hotta et al. 2005). At a later stage of neuronal development, CRMP-5 is thought to regulate dendritic development by mediating brain-derived neurotrophic factor signaling in the CNS (Yamashita et al. 2011). CRMP-5 competes with CRMP-2 for interaction with tubulin dimers, thus impairing tubulin transport and subsequently arresting the tubulin polymerization and neurite outgrowth (Brot et al. 2010). CRMP-5 and CRMP-1 are also shown to have antagonistic effects, with CRMP-5 negatively regulating and CRMP-1 stimulating the proliferation of neural progenitors and transit amplifying neuroblasts (Veyrac et al. 2011). Moreover, CRMP-1 and CRMP-5 are thought to contribute synergistically to the regulation of the balance between functional integration and death of newborn neurons in the adult neurogenic areas, via pathways that exclude direct molecular interaction between these two proteins (Fukada et al. 2000; Su et al. 2007; Veyrac et al. 2011).
CRMPs preferably occur as hetero-tetramers in vivo, but form homo-tetramers when purified from bovine brain (Wang and Strittmatter 1997). The core region, which excludes the C-terminal approx 80 residues of CRMPs, is hereby sufficient for the oligomerization (Wang and Strittmatter 1997; Stenmark et al. 2007; Majava et al. 2008). Divalent cations such as Ca2+ and Mg2+ have been reported to promote homo-tetramer formation of CRMP-2 in solution (Majava et al. 2008), whereas in the crystalline state, murine CRMP-1 (15–490) as well as human CRMP-2 are homo-tetrameric even in absence of divalent cations (Deo et al. 2004; Stenmark et al. 2007). In solution, CRMP-5 was shown to form hetero-oligomers with CRMP-2, -3, and -4, but interestingly not with CRMP-1 (Fukada et al. 2000).
Compared with their closest homolog, the enzyme DHPase, the sequence of CRMPs, is extended by a positively charged C-terminal region, which is highly susceptible to proteolysis (Deo et al. 2004 and R.P. unpublished results). In vivo, the calcium-activated protease, calpain, specifically cleaves CRMPs within the C-terminal region to produce 55- to 58 kDa products (Jiang et al. 2007). These truncated proteins have various effects on axonal growth and sometimes even induce cell death after translocation into the nucleus (Jiang et al. 2007; Zhang et al. 2007; Taghian et al. 2012). However, little is known about the direct effects of the C-terminal truncation on structure and function of CRMPs. Knowledge of the structure and oligomerization properties of CRMP-5 in comparison with other members of the CRMP family is thus required to understand its unique role.
Here, we report the crystal structures of the full-length (WT) and a truncated version (ΔC) of human CRMP-5 at 2.2 and 1.7 Å resolution, respectively. For the former, only residues 1–491 are visible in the electron density map, but a possible location of the C-terminal region can be identified. Comparison of the active site of DHPase with the corresponding site of CRMP-5 reveals small, but crucial changes that explain the lack of amidohydrolase activity. As expected, the overall structure of CRMP-5 is very similar to that of CRMP-1 and CRMP-2. Nevertheless, the observed differences allow conclusions about why CRMP-5 preferentially forms hetero-oligomers with CRMP-2 rather than CRMP-1. Analysis of the oligomerization states of CRMP-1, -2, and -5 at varying protein concentrations and in absence or presence of divalent cations revealed significant differences, which, in addition to protein phosphorylation/dephosphorylation events may be crucial for regulation of CRMP function and interaction with other signaling and cytoskeleton proteins. Such a regulation via oligomerization has already been described for CRMP-2, which dissociates into monomers when interacting with tubulin and calmodulin (Fukata et al. 2002; Zhang et al. 2009).
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- Materials and methods
- Conflict of interest
- Supporting Information
We have determined the crystal structures of WT and a C-terminally truncated form of human CRMP-5, the latest identified member of the CRMP protein family. CRMP-5 crystallizes as a homo-tetramer similar to that previously described for CRMP-1 and CRMP-2 (Deo et al. 2004; Stenmark et al. 2007). Analysis using gel filtration revealed that, in contrast to CRMP-2 (Wang and Strittmatter 1997; Stenmark et al. 2007; Majava et al. 2008), CRMP-5 and CRMP-1 form relatively weakly associated homo-tetramers in solution (Fig. 4). However, all CRMPs were shown to have a preference for formation of hetero-tetramers over homo-tetramers in vivo, with the N-terminal core region (8-134 and 281-435) being essential and sufficient for the assembly of this oligomerization state (Wang and Strittmatter 1997; Fukada et al. 2000). On the basis of the existing and presented structural data, we identified residues at both the arm–arm and arm–lever dimer interfaces, which in the homo-oligomer are less ideally positioned or interacting, and may thus play a role in the discrimination between homo-and hetero-oligomerization states. Furthermore, we also identified an interaction hotspot in both the arm–arm and arm–lever dimer interfaces that could explain why CRMP-5 can form hetero-tetramers with all CRMPs except CRMP-1 (Fukada et al. 2000). Both are characterized by changes of positively charged (K265 and K474) to uncharged residues (Pro and Gln, respectively). In addition, we showed that CRMP-5 and CRMP-1 have a lower propensity to form homo-tetramers than CRMP-2, which may indicate that formation of hetero-tetramers is not favored either. Based on the available structural data, it is not possible to exclude that more than two different CRMPs constitute one hetero-tetramer, which would allow CRMP-5 and CRMP-1 to be present in one tetramer as diametrically opposed monomers that do not interact. Similarly, it was proposed earlier that the presence of multiple combinations of CRMP hetero-oligomers in a neuron is necessary for a functional neuronal network (Fukada et al. 2000; Deo et al. 2004). Nonetheless, the precise manner in which CRMP hetero-tetramers are formed, that is, whether by using arm–arm or arm–lever homo-dimers, is still unclear. Stenmark et al. proposed a CRMP-1–CRMP-2 hetero-tetramer model via the arm–arm interface using arm–lever-interfaced homo-dimers owing to the higher degree of sequence conservation (Stenmark et al. 2007). However, the arm–lever interface in CRMP-1–CRMP-2 shows a larger contribution from hydrophobic residues compared with the arm–arm interface. Unlike the CRMP-1–CRMP-2 hetero-tetramer analyses, other hetero-oligomer analyses do not show any obvious discrimination between the two interfaces. Therefore, it cannot be ruled out that the arm–lever interface is also used in the hetero-tetramer assembly, especially as there are residue pairs in both interfaces that seem to favor the hetero-tetramer. The functional importance of CRMP homo- and/or hetero-oligomerization is still unclear. One possibility would be that hetero-tetramer formation would bring different C-terminal regions into close proximity. These regions are least conserved (see supporting Table 2) and may allow CRMPs to interact with the same or different partners in a precise and fine-tuned manner (see below). Thus far, two in vivo studies reported a modulation of axon growth in correlation to the oligomerization of CRMPs. In one study, the e204 mutation in the unc-33 gene from C. elegans (residue D58 is mutated to Asn) causes paralysis resulting from defective axon growth (Brenner 1974). The D58N mutation hampers the oligomerization of UNC-33 and its binding with UNC-14 (RUN domain protein) and Kinesin Light Chain-2 (Tsuboi et al. 2005). D58 is conserved between CRMPs and UNC-33 (D64 in CRMP-5; D71 in CRMPs 1-4). In CRMP-2, the D71N mutant does not interfere with the oligomerization of the protein, but specifically with the interaction to Sra-1 (Rac-1 associated protein 1). Nevertheless, this also results in axon growth defects (Kawano et al. 2005). The corresponding residue D64 is located in the core of CRMP-5, in fact exactly at the center of the mass of monomer molecule, and hence distant to both oligomerization interfaces (Figure S5). The effects of the mutation seen in UNC-33 and CRMP-2 may not be based on a direct interference with the oligomerization, but an indirect effect caused by the destabilization of the monomer core. In the second study, alanine substitution mutants of residues 367-368 and 487-489 of mouse CRMP-1 produced functionally inactive proteins, even though both retained their ability to interact with plexin A1. The residues within the mutated region appear to be crucial as they are conserved among CRMPs. They are located in the vicinity of the arm–arm interface and might destabilize the functional tetramer (Deo et al. 2004) (Figure S5).
The divalent cations Ca2+ and Mg2+ were shown to promote CRMP-2 oligomerization in solution (Majava et al. 2008) and to bind to CRMP-2 in crystals (Stenmark et al. 2007; Majava et al. 2008). The Ca2+-and Mg2+-binding sites identified in the corresponding CRMP-2 crystal structures overlap and are found on the tetramer surface distant from dimer interfaces. It is therefore not directly evident how these cations promote oligomerization. Also, both cations appear to be highly coordinated by water molecules that mediate the contact to the protein. In particular, the Ca2+ ion is bound directly to the main chain carbonyl oxygen of residue 349, and indirectly via water molecules to the side chains of E353 or Q81 of CRMP-2. While these latter residues are conserved in CRMP-1, in CRMP-5, they are replaced by H346 and M71, respectively, which do not possess the size or charge to interact similarly with these cations. Later, Majava et al. reported two additional Ca2+-binding sites in CRMP-2, but the data have not yet been deposited in the protein data bank (PDB) (Majava et al. 2008). Although these sites are not directly involved in the tetramer interface formation, the site near the side chain of Q245 belonging to α-helix 7 possibly stabilizes the CRMP-2 monomer and thereby assists in the tetramer formation. Q245 is replaced by arginine in CRMP-5 and CRMP-1, making it an unlikely binding site in these two proteins. In line with this structural evidence, CRMP-5 and CRMP-1 oligomerization was not promoted by Ca2+ or Mg2+ in vitro (Fig. 5), and the thermal stability of both proteins was decreased rather than increased in the presence of these ions (Fig. 4). In vivo, both cations are important in cellular signaling and development, and especially Ca2+ has an important role in axon guidance. Several proteins and functions connect calcium ions directly to CRMPs. For example, CRMP-2 interacts with calcium channels and calmodulin, and calpain is activated by the ion (Zhang et al. 2007, 2009; Brittain et al. 2009). However, the in vivo relevance and the functional role of divalent cation binding to CRMPs are still to be investigated.
Metal binding and enzymatic activity of CRMP-5
As CRMP-5 is the closest relative to the enzyme DHPase within the CRMP family, it could be expected to have some residual DHPase activity. However, several residues in the active site, including the carboxylated lysine residue that coordinates the catalytic metal center in DHPase, are exchanged in CRMP-5, which effectively abolishes metal binding as shown by mutational analyses of hydantoinases (Huang et al. 2009). This was confirmed experimentally and consequently no residual amidohydrolase activity of CRMP-5 is detectable. Chemical rescue by small carboxylic acids that could take over the role of the carboxylated lysine in zinc binding is probably hindered sterically and electrostatically by the presence of the glutamine side chain at the equivalent position near the binuclear metal center site. Substrate binding is most likely affected as well, even though no side-chain-specific substrate interactions are made in DHPase (Lohkamp et al. 2006). In CRMP-5, R327 is protruding into the ‘active site’ cavity, partially occupying the substrate-binding site. In addition, its positive charge may interfere with zinc ion binding. It thus appears that evolution to a non-enzymatic entity with alternative functions in neuronal development is already completed in CRMP-5, and that it does not represent a ‘missing link’ between the enzymatic and non-enzymatic forms. The likelihood that CRMPs show enzymatic activity toward yet unidentified other substrates is low as this would require the evolution of a different, metal-independent catalytic machinery.
Whether DHPases can substitute CRMP function, or has roles distinct from those of CRMPs, in neuronal development remains to be seen. Interestingly, deficiency in DHPase activity is associated with neurological abnormalities (Henderson et al. 1993), although the mechanisms involved may not be connected to CRMPs.
Structure and function of the C-terminal domain of CRMP-5
Similar to previous studies, we were not able to resolve the full-length structure of a CRMP. Although we crystallized the full-length CRMP-5 protein, only residues up to 492 were visible in the experimental electron density map. The missing electron density for the remainder of the protein may be attributed to its unstructured character, in other words flexibility and lack of permanent secondary structure. FoldIndex predicts the C-terminal region to be intrinsically unfolded (Prilusky et al. 2005). Furthermore, while the theoretical isoelectric point of CRMP-5 WT is 6.7, this value increases to 10.14 for the C-terminal region alone (last 84 amino acids), rendering it highly basic. For the crystal structures of CRMP-1 and -2, C-terminal residues were not even included in the structure determination because of their proteolytic susceptibility, and thus no structural information is available (Deo et al. 2004; Stenmark et al. 2007). Interestingly, the resolved CRMP-5 residues in the presented structures correspond almost exactly to two putative calpain-cleaved products, indicating their biological relevance (Taghian et al. 2012). Based on the CRMP-5 WT structure, it can be postulated that the C-terminus is likely to extend away from its own protomer. Residues 483-492 do not contribute much to the arm–lever dimer interface, with only one salt bridge being formed by R489 from the end of the extended loop. Mutation of this residue to alanine in a bacterial DHPase was shown to have no effect on the tetramer assembly (Martínez-Rodríguez et al. 2010). The remainder of the C-terminal region of CRMPs harbors many functionally important residues such as the phosphorylation sites crucial for regulating CRMPs' interactions with various binding partners, and a KKEX motif (526-529), which is commonly present in type I microtubule-associated proteins (Noble et al. 1989). Tubulin was shown to bind near the C-terminus of CRMP-5, specifically to the region 475-522 (Brot et al. 2010). As most, if not all, of the phosphorylation sites in CRMP-5 are found in the C-terminal region, it is likely that tubulin binding to CRMP-5 is regulated by phosphorylation. This is, for example, observed in the protein stathmin, which also interacts with tubulin dimers in a phosphorylation-dependent manner (Charbaut et al. 2001) and shows a low sequence similarity to CRMP-5. However, it appears that the C-terminal domain is not directly involved in binding to tubulin as surface plasmon resonance experiments indicated a low μM affinity of CRMP-5 ΔC for tubulin, which is comparable with that of the WT protein and other CRMPs (R.P. unpublished result, Fukata et al. 2002). Interaction with tubulin or other proteins at the C-terminus is unlikely to affect the structure of the core protein, thereby changing binding properties and function. However, it is not possible to rule out that the C-terminus interferes with binding epitopes on the core of the CRMP, thus modulating interaction of CRMP with other proteins.
CRMPs are regulatory phosphoproteins abundantly present in the early stages of neuronal development. Their spatiotemporal expression pattern varies between the members of the family. As a hub protein in a complex network of protein–protein interactions, CRMPs have to be highly regulated. This appears to be mainly achieved by phosphorylation; however, there is growing evidence that oligomerization may play a role as well. The formation of homo- and/or hetero-oligomers is imperative for CRMPs function in vivo, but their nature of composition during the various stages of neuronal development remains elusive. Availability of structural details of CRMP-1, -2, and -5 at atomic level will help to elucidate the in vivo oligomerization effect on CRMP complexes with other proteins by assisting a targeted mutagenesis approach with the aim to stabilize or destabilize specific homo- or hetero-tetramers.