Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect

Abstract

Charcot–Marie–Tooth (CMT) diseases are the most common heritable peripheral neuropathy. At least 10 different mutant alleles of GARS (the gene for glycyl-tRNA synthetase) have been reported to cause a dominant axonal form of CMT (type 2D). A unifying connection between these mutations and CMT has been unclear. Here, mapping mutations onto the recently determined crystal structure of human GlyRS showed them within a band encompassing both sides of the dimer interface, with two CMT-causing mutations being at sites that are complementary partners of a “kissing” contact across the dimer interface. The CMT phenotype is shown here to not correlate with aminoacylation activity. However, most mutations affect dimer formation (to enhance or weaken). Seven CMT-causing variants and the wild-type protein were expressed in transfected neuroblastoma cells that sprout primitive neurites. Wild-type GlyRS distributed into the nascent neurites and was associated with normal neurite sprouting. In contrast, all mutant proteins were distribution-defective. Thus, CMT-causing mutations of GlyRS share a common defect in localization. This defect may be connected in some way to a change in the surfaces at the dimer interface.

Dominant mutations in human glycyl-tRNA synthetase (GlyRS) (1–6) and tyrosyl-tRNA synthetase (TyrRS) (7) are amongst the genetic causes of Charcot–Marie–Tooth (CMT) disease, the most common heritable peripheral neuropathy (8). The rationale for two members of the aminoacyl tRNA synthetase family of proteins being associated with CMT is not known. One hypothesis is that, in a way that is not understood, defects in translation lead to the disease phenotype (5). Indeed, in the case of TyrRS and GlyRS, activities of specific CMT-causing mutant GlyRSs and TyrRSs were reported to be deficient (5, 7). In contrast, however, in a mouse model of CMT, the disease-causing mutant GlyRS is fully active for aminoacylation (4). In addition, a mouse heterozygous for a loss-of-function Gars allele has a normal phenotype, even though the level of GlyRS activity in cell lysates is reduced by the expected two-fold (4). This observation has turned attention to the possibility that an alternative function of GlyRS and TyrRS, associated with neuronal homeostasis or development, is behind the CMT-connection. This possibility has been fostered by the growing awareness of the expanded functions of specific human tRNA synthetases, which appear to link translation to the systems biology of broad signaling pathways in higher organisms (9).

In the case of the homodimeric human GlyRS, at least 10 dominant mutations have been annotated (1–6). The mutations do not cluster together and, instead, scatter across the sequence in a way that suggests no obvious relationship between them. However, the recent determination of the 3D structure of human GlyRS affords an opportunity to now examine the spatial relationships between the sites of the mutations, and to see whether those relationships suggested a unifying theme. In that connection, a recent structure, and a functional analysis, of one mutant protein showed that the dimer interface was sensitive to a CMT-causing mutation that was itself distal to that interface (10). This observation raised the possibility of interconnections within the structure of GlyRS that could, in principle, provide a rationale for the scattered locations of the various mutations that caused CMT. For example, we wanted to see how the mutations were positioned relative to the dimer interface. If those locations suggested the possibility of mutational effects on the interface, then that would provide motivation to examine experimentally the dimerization interaction. At the same time, the structure also gave us the opportunity to model and to understand the locations of the mutations relative to the active site and the tRNA binding interface. This information could provide the foundation and rationale for studying in more detail the relationship, if any, between disease and aminoacylation activity. Because TyrRS distributed strongly into sprouting neurites of neuroblastoma cells, and this selective localization is lost with mutant forms of TyrRS (7), we wanted to investigate GlyRS for the same phenomenon. The rationale was that if a neurite distribution pattern similar to that of TyrRS was seen, then effects of mutations on that distribution pattern might unify the various mutant proteins, and do so in a way that could relate to the dimerization interface or aminoacylation activity.

Results

Mapping of CMT-Causing Mutations.

Human GlyRS is a homodimer with the monomer unit having 685 residues composed of an N-terminal appended WHEP-TRS domain (disordered in the crystal structure), a catalytic domain, and a C-terminal anticodon binding domain (10). The catalytic domain contains the characteristic three conserved motifs (1, 2, and 3) of class II tRNA synthetases and, in addition, three insertions (I, II, and III) between the motifs. The 10 reported CMT-causing mutations are spread throughout the primary sequence of human GlyRS. (In the description below, residues at positions associated with CMT-causing mutations are put in italic font. Residues on opposite subunits are distinguished by unprimed and primed designations.) When these mutations are placed on the structure, all of them concentrate around a band that is centered on the dimer interface (Fig. 1A). Eight (E71G, L129P, P234KY, G240R, I280F, H418R, D500N, G526R) are in the catalytic, and two (S581L and G598A) are in the anticodon binding domain (Fig. 1B). Except for D500N, all are within the conserved regions shared by human and T. thermophilus GlyRS. Interestingly, D500N, which lies in the disordered insertion III, is the only CMT-causing mutation that is not resolved in the crystal structure.

Fig. 1.

Mapping of CMT-causing mutation sites on the crystal structure of human GlyRS. Residues on opposite subunits are distinguished by unprimed and primed designations. (A) Nine resolved CMT residues are displayed on each subunit of the dimeric GlyRS. The two subunits are colored in beige and green, respectively. All CMT-associated positions are concentrated in a band around the dimer interface. (B) The green subunit in A is rotated 90° along the x axis to shown its dimer interface. The color of the subunit is changed to show the different domains, insertions, and motifs. The catalytic and anticodon binding domains are in yellow and green, respectively. Insertions I and II and motifs 1, 2, and 3 are in cyan and red, and in magenta, pink and orange, respectively, on one subunit. All CMT-associated residues are shown and colored in blue for this subunit, or colored in red for the other subunit. (Inset) The interaction of CMT-associated S581 with H591. (C) The five CMT-associated residues that make dimer interactions, including a kissing interaction between two of them (P234 and I280′), across the dimer interface.

Most Catalytic-Domain Mutations Are at the Dimer Interface, Including a “Kissing” Contact Between Two CMT Mutation Sites.

Except for G526 (in the active site for adenylate synthesis (10) and E71 (close to the N terminus of the catalytic domain), five of seven resolved CMT-causing mutations in the catalytic domain are involved in interactions that bridge the dimer interface. Interestingly, these five residues concentrate on one side of the dimer interface, which is designated as the “front” of the dimer shown in Fig. 1A. A subunit exposing the dimerization interface is shown in Fig. 1B, which is related to the bottom subunit of the dimer (shown in Fig. 1A) by 90° rotation along the x axis, so the front of Fig. 1A becomes the bottom of Fig. 1B. Fig. 1C shows the specific interactions at the dimer interface made by these five residues. Specifically, the backbone N of L129, a motif 1 residue, forms an H-bond with the side chain carboxylate of E291′ from the other subunit. G240 on strand β9 interacts, through T230 on β8, with K229′ from the other subunit. (Interestingly, L129 is <7 Å away from G240.) Also, the side chain imidazole ring of H418 on α12 forms an H-bond with the Y87′ side chain OH from across the dimer interface.

Most interestingly, P234 and I280 (each at a position associated with a CMT-causing mutation) make kissing contact across the dimer interface (Fig. 1C): P234, which is at the loop between β8 and β9 and in the sequence a few residues before the above-mentioned G240, from one subunit interacts with I280′, which is a motif 2 residue located on loop between β10 and β11, of the other subunit. The distance between the two residues is <4 Å. In addition to this remarkable CMT-associated kissing contact, the clear concentration of CMT-mutations at the dimer interface, and especially at the front side of the interface, suggest strongly the importance of that interface for the mechanism of CMT.

CMT-Causing Mutations in the Anticodon Binding Domain.

Two CMT-associated mutations, S581L and G598A, are in the anticodon-binding domain and are not directly involved in dimerization. S581 is located on helix α14 and has specific side chain interactions to another anticodon-binding domain residue (H591) within the same subunit (Fig. 1B Inset). The S581L mutation would disrupt this interaction. Although S581 and the overall anticodon-binding domain do not make dimer interface contacts, S581 is part of the “loose” dimer contact area that is strengthened by G526R, a CMT-causing mutation in the active site ≈30 Å away (10). The other CMT-associated position, G598, precedes helix α15 and, in contrast to S581, is solvent-exposed and free of interactions (Fig. 1B).

Most Tested CMT-Causing Mutations Affect Dimer Formation.

The concentration of CMT-causing mutations in a band centered on the dimer interface directs attention to this interface and the possibility that it plays a key role in explaining the connection of CMT to the distribution of mutations. [Interestingly, in an investigation of granule formation in mouse motor neuron cells by Antonellis et al. (5), we noted that, in the light of our structure, mutants that did not form granules (L129P, G240R, H418R) are all at the dimer interface, and ones that maintained granules are not at the interface.] We described an experimental system to study effects of mutations on dimerization of GlyRS (10). In this system, genes encoding V5-tagged WT and mutant human GlyRS were transfected into mouse neuroblastoma N2a cells. (These cells also expressed endogenous mouse GlyRS from the chromosome.) After 24 h, cells were lysed and immunoprecipitated with anti-V5 antibodies to pull-down the GlyRSs that were expressed from the recombinant, transfected genes. The redissolved precipitates were then subjected to SDS/PAGE and Western blot analysis with polyclonal antibodies directed against all forms of GlyRS. The idea is that, by using anti-V5 antibodies for the pulldown and the anti-GlyRS antibodies for the Western blot analysis, any native GlyRS that “hybridized” in vivo with V5-tagged GlyRS would be detected. The hybridization is possible to detect because of the extra mass of the V5-tagged protein that enables it to be distinguished from the native enzyme. Thus, the “in vivo” experiment was designed to look at how CMT-causing mutations affect heterodimer formation between CMT-causing mutant proteins and WT GlyRS. This kind of approach is of particular interest because all CMT-causing mutations are dominant, that is, they express their effects when one GARS allele is mutant, whereas the other is the WT.

We investigated 7 of the 10 CMT-causing mutations for their ability to form heterodimers with the WT mouse GlyRS (95% sequence identity to human GlyRS) (Fig. 2A). (Among those, the mutant enzyme, G526R, was described in ref. 10.) As a reference point, when cells were transfected with V5-tagged WT GlyRS, ≈14% of the transfected GlyRS was in the form of the heterodimer. This number was little changed by the P234KY or H418R mutations. However, the other five tested mutant genes expressed proteins that affected dimer formation in one way or another. In particular, D500N, G526R, and S581L mutations strengthen the capacity to form heterodimers with 34%, 35%, and 47% of the transfected GlyRS in the form of the heterodimer, respectively. In contrast, the L129P and G240R substitutions yield proteins that showed no evidence of heterodimer formation.

Fig. 2.

Effect of CMT-causing mutations on dimer formation. (A) Immunoprecipitation experiments showing that V5-tagged mutant GlyRSs “pulled down” various amounts of endogenous GlyRS, presumably by forming dimers. L129P and G240R GlyRS did not pull down any endogenous GlyRS, whereas D500N, G526R, and S581L GlyRSs pull down more endogenous GlyRS than did WT GlyRS. P234KY and H418R GlyRSs pulled down endogenous GlyRS in an amount similar to that seen with WT GlyRS. (B) Analytical ultracentrifugation experiment showing G240R GlyRS exists mainly as a monomer, whereas more dimers are formed by G526R mutant than by WT GlyRS (Inset).

To validate the results we obtained from the cell-based in vivo system, we picked two mutations, G240R and G526R. One is representative of disrupted heterodimers (G240R), whereas the other had strengthened interactions (G526R), according to the above-described in vivo system. These two were studied by analytical ultracentrifugation along with WT GlyRS. The result of ultracentrifugation analysis for G526R GlyRS was described in ref. 10 and confirmed that the G526R substitution enhances homodimer interaction (Fig. 2B Inset). In contrast, a major peak (S value ≈5.0) and a small broad peak (S value ≈7.0) was observed by sedimentation velocity analysis for G240R GlyRS (Fig. 2B), suggesting G240R GlyRS exists mainly as a monomer in the ultracentrifugation experiment. Thus, with both analyses used to study subunit interactions, the same conclusions were reached.

From the structural perspective, it is not surprising that L129P and G240R mutations disrupted dimer formation because, as shown in Fig. 1C, both L129 and G240 are involved with dimerization contacts as internal residues having constraints that cause conformational rigidity and restriction. In contrast, P234 and H418, although interacting with residues from the other subunit, have a surface, rather than an internal, location and, therefore, are not as likely to affect the stability of the dimer. In particular, P234 is located on an exposed and potentially flexible loop (between strands β8 and β9). Its kissing interaction with I280′ across the dimer interface may be maintained in a different way with the P234KY mutation. H418 is located just before the disordered insertion III, and its cross-dimer partner Y87′ is located on a long loop in between two β- strands (β1 and β2). Given the flexibility of this region, the H418R mutation may not disrupt the cross-dimer interaction.

It is of particular interest that the three mutations that enhanced dimer formation (D500N, G526R, and S581L) are more distal to the dimer interface. This observation, made originally in studying the effects of the G526R mutant protein (10), is reinforced now by the new results with the D500N and S581L mutant enzymes. Thus, long-range conformational communication in human GlyRS transports a local disturbance to a distant place. This long-range communication may explain, at least in part, why, within the band (centered on the dimer interface) where they are concentrated, CMT-causing mutations can perpetrate their effects from different positions.

CMT-Causing Mutations That Potentially Affect tRNA Interaction.

We sought to understand whether the CMT-causing mutations would affect the interaction of GlyRS with its tRNA substrate. Pursuant to this objective, we generated a model for the tRNA complex. Among the several complexes of class II tRNA synthetases that have been solved with bound tRNA, E. coli ThrRS has the conformation most close to our structure of human GlyRS. Therefore, using the E. coli ThrRS-tRNA^Thr complex (11) [Protein Data Bank (PDB) ID code 1QF6], we superimposed the two catalytic domains (one from ThrRS and one from human GlyRS) to generate a model for GlyRS bound to tRNA (Fig. 3A). In this model, the tRNA fits onto the structure of human GlyRS, with the anticodon docking to the anticodon binding domain, and the 3′-CCA acceptor terminus fitting into the active site. However, because insertion I makes some steric clashes with the acceptor stem at the 5′ end of the docked tRNA, the conformation (or the orientation relative to the rest of the enzyme) of insertion I would most probably be modified when tRNA is bound. Easily, insertion I could make a conformational change, because it is structurally separated from the catalytic domain, and is away from the dimer interface (10). In the crystal, insertion I is making lattice contacts with the anticodon recognition domain, suggesting that the current conformation of insertion I could be somewhat influenced by crystal lattice interactions. In this connection, insertion I was completely disordered in the crystal structure of T. Thermophilus GlyRS (12, 13), presumably because of lack of both tRNA binding and crystal lattice interactions.

Fig. 3.

CMT residues that have potential tRNA interactions and the effect of CMT-causing mutations on tRNA aminoacylation. (A) Stereoview of the model of tRNA in a complex with GlyRS. Four of the nine resolved CMT-causing mutations are close to the tRNA interface: they are E71, P234′, I280, and G598. (B) Aminoacylation assay performed with the WT and seven mutant GlyRSs. Four different mutants of GlyRS (E71G, P234KY, D500N, and S581L) had WT-like activity, whereas three others (L129P, G240R, and G526R) were inactive.

Four of the nine resolved CMT-causing mutations are close to the tRNA interface. These are E71, P234, I280, and G598 (Fig. 3A). [Also, the lone CMT-causing mutation (D500) that is located in the disordered region of insertion III can potentially be involved in tRNA recognition (10).] E71 lies inside the elbow of the L-shaped tRNA, and potentially makes contact with both the acceptor- and D-stems. G598 is close to the 5′-side of the anticodon. Interestingly, some of the tRNA interface is proximal to the dimer interface. In this connection, the kissing residues P234 and I280′ at the dimer interface also are located close to the 5′-end of the bound tRNA. Thus, in principle, mutations at positions 234 and 280 could affect either or both of a dimer or tRNA interaction.

No Correlation of CMT-Causing Mutations with Aminoacylation Activity.

As stated in Introduction, no clear picture has emerged for a connection between aminoacylation activity and CMT. In the mouse model of CMT, the P278KY mutation (equivalent position as P234 in human GlyRS) had no effect on aminoacylation activity (4). In contrast, G526R GlyRS has abolished aminoacylation activity (10), supporting other reports suggesting a connection between aminoacylation activity that acts through effects on protein synthesis (5). Given these circumstances and considering that the structural analysis above suggested a possible connection of at least some CMT-causing mutations to tRNA interactions, we investigated the aminoacylation activities of additional mutant enzymes.

We investigated three of the five mutant enzymes that have substitutions potentially close to the tRNA interface. These were E71G and D500N GlyRS, and P234KY GlyRS that were characterized in ref. 4. All three of them were fully active for aminoacylation (Fig. 3B). These results reinforce the idea that aminoacylation per se is not a fundamental cause of CMT. In addition, the results suggest that E71, P234, and D500 do not have an essential (for aminoacylation) interaction with tRNA.

To investigate further the question of the role of aminoacylation in CMT, we also studied L129P, G240R, and H418R GlyRS, as examples where the WT residue is involved with dimerization. Of these three mutant enzymes, L129P and G240R GlyRS was completely inactive (Fig. 3B), whereas H418R GlyRS had some residual aminoacylation activity (data not shown). Because the dimeric form of GlyRS is essential for aminoacylation activity (14), the loss of aminoacylation activity for L129P and G240R is likely due to disruption of the dimer.

Of the two anticodon-binding domain mutant enzymes (S581L and G598A) we studied S581L GlyRS and found it was fully active for aminoacylation (Fig. 3B). Thus, including our previously studied example of the inactive G526R GlyRS (10) and the active murine P234KY GlyRS (4), eight mutant enzymes have been investigated. Of these eight, half are active. These results support strongly the conclusion that CMT is not caused by defects in aminoacylation.

GlyRS Distributes into Neurite Projections of Differentiating Neuroblastoma Cells.

We wished to determine whether GlyRS distributes to sprouting neurites, as has been observed with TyrRS (7), the other tRNA synthetase associated with CMT. Immunofluorescence experiments by Jordanova et al. demonstrated different subcellular distributions of TyrRS in neuronal and nonneuronal cells. In differentiating mouse (N2a) and human (SH-SY5Y) neuroblastoma cells, endogenous TyrRS concentrated in granular structures in the growth cone, branch points, and the most distal part of projecting neurites. This phenomenon was called the “tear-drop effect”. It was observed in all cells with neurite projections. Further work with anti-TyrRS immunofluorescence-staining in primary embryonic (E14) motor neurons confirmed that the protein localized not only throughout the cell body, but also in the distal part of the smallest filopodia projecting from the main neurite (7). Importantly, these experiments confirmed that the “tear-drop” distribution of TyrRS could be seen in the whole organism and not just neuronal cells in culture.

We adapted this system to see whether GlyRS also distributed into the distal parts of sprouting neurites of N2a neuroblastoma cells that were transfected with genes encoding V5-tagged WT human GlyRS. Of course, the N2a cells also express the endogenous WT mouse GlyRS from the chromosome. This circumstance gave us the opportunity to investigate separately the distribution of both endogenous and transfected proteins, using different immunofluorescence detection systems.

Immunofluorescence with anti-GlyRS antibodies, which detect both the endogenous and the transfected GlyRS, showed a clear distribution of GlyRS in the cell body and distal parts of the sprouting neurites (Fig. 4 top left). A similar distribution was seen by using anti-V5 antibodies, which only detect the transfected GlyRS (Fig. 4, top right). We compared the intensity of fluorescence in the neurite verses that in the cell body, and found that the relative intensity was the same for the two images one stained with anti-GlyRS antibodies and the other with anti-V5 antibodies. This result shows that the transfected V5-tagged WT GlyRS distributes to the sprouting neurite as well as endogenous GlyRS. Thus, GlyRS is distributed throughout the sprouting neurite.

Fig. 4.

Immunofluorescence study in mouse neuroblastoma N2a cells showing that all CMT-causing mutants are defective in neurite localization. Cells transfected with genes encoding WT or mutant GlyRSs are stained with anti-GlyRS antibody and with anti-V5 antibody, to investigate both endogenous and transfected proteins, respectively. Each antibody-staining image has two arrows, one pointing to the cell body, the other to a sprouting neurite. The relative fluorescence intensity of the neurite to the cell body was compared between the two different antibody stainings, and the ratio was calculated.

All Investigated CMT-Causing Mutants Are Defective in Their Localization to Neurite Projections.

Having established the localization of GlyRS to developing neurites, we investigated seven mutant GlyRSs (L129P, P234KY, G240R, H418R, D500N, G526R, and S581L) in experiments similar to those described above. Each of the seven CMT-causing mutants was transfected into N2a neuroblastoma cells as V5-tagged fusion proteins. Remarkably, all seven mutant GlyRSs were defective in their localization to sprouting neuritis [Fig. 4 Right (except the top panel)], where the endogenous GlyRS was still maintained [Fig. 4 Left (except the top panel)]. To quantify the defect, the relative fluorescence intensity of the neurite to the cell body was compared between the two images, one that visualized both the endogenous and the transfected GlyRS, and the other that visualized only the transfected mutant GlyRS.

Compared with the endogenous GlyRS, and in the cells picked and shown here, L129P and P234KY GlyRS had a >10-fold decrease in neurite distribution. G240R, H418R, D500N, and G526R GlyRSs had 5- to 10-fold decreases, whereas S581L GlyRS had a ≈2-fold decrease. Thus, although there is variability from cell to cell and mutant to mutant, all of the seven tested CMT-causing mutant enzyme had diminished or defective distributions into the nascent neurite projection.

Discussion

The broad distribution of CMT-causing mutations on the primary sequence of GlyRS has long been puzzling. However, with our recently solved crystal structure, the apparent dispersion of the mutations is diminished substantially. In particular, all nine CMT-associated positions, where the residues could be resolved in the structure, are proximal to the dimerization interface and, in addition, five are involved with subunit interactions across the dimer interface. However, the effects of the CMT-causing mutations on dimer formation in vivo and in vitro show a distribution of effects, ranging from stronger dimer interactions (D500N, G526R, and S581L GlyRS) to completely disrupted subunit interactions (L129P and G240R GlyRS).

L129P and G240R GlyRS are stable as monomers and could be detected in transfected N2a neuroblastoma cells and purified as recombinant proteins from bacteria. Thus, the monomer unit itself can fold and form a stable structure, without need for the dimer interaction for stabilization. The stand-alone monomers may have a different, and maybe less tight, conformation than those in the dimer form, and the altered conformation could be the general cause for the lost aminoacylation activity of the monomers. Interestingly, the homodimer of WT GlyRS is in a dynamic equilibrium with the monomer (Fig. 2B), suggesting that the monomer form is normally present at some level. Because the monomer is inactive for aminoacylation, a distinct biological role could be considered for the monomer.

All CMT-causing mutations in GARS have a dominant phenotype, which is typically associated with a gain-of-function. Heterodimer formation between a WT and mutant subunit is one way to acquire a new (in this case, pathological) function. However, because L129P and G240R mutant proteins are monomeric, they have less capacity to form heterodimers with the WT subunit and thereby “poison” aminoacylation or any other activity associated specifically with the dimer. Indeed, such heterodimers were not detected in lysates of N2a cells transfected with genes for the L129P and G240R mutant proteins (Fig. 2A). Related to this point, the CMT-associated D500N and S581L GlyRSs have stronger dimer interactions and can form heterodimers. And yet, these mutant proteins are fully active for aminoacylation as homodimers and, therefore, are unlikely to have the ability to poison the WT protein, at least with respect to the aminoacylation activity. Thus, our data provide no support for the idea that subunit poisoning is causally connected to CMT arising from mutations in GlyRS.

Regarding the dimerization interface, most striking is the kissing interaction between P234 and I280 and the association of CMT with a mutation of either of these residues. The a priori statistical likelihood that “complementary” residues could randomly appear as sites of mutations for CMT is small. Therefore, that two residues make cross-subunit contacts, with each being the locus of a CMT-causing mutation, is powerful circumstantial evidence for a connection between CMT and the dimerization interface. Moreover, additional interconnections between CMT-causing mutations can be seen. As an example, G526R has a long-range structural effect on another CMT-associated residue, i.e., S581. As a result of this long-range effect, G526R GlyRS has a stronger dimer interaction (10). Interestingly, substituting the hydrophobic leucine for the hydrophilic serine, to give the CMT-causing S581L GlyRS, also provides a stronger dimer interaction (Fig. 2A). Thus, whether one considers direct close interactions, as seen in the kissing contacts, or whether indirect connections linking residues that are further apart are considered, a connection of CMT to the dimer interface is made.

The precise link between CMT and the dimerization interface is not clear, however. The interface region of the dimer may be critically linked to CMT and, simultaneously or alternatively, the interface itself (which is exposed in the monomer) may be the critical feature. Regardless, all 5 CMT-associated positions that have residues making dimer interactions are clustered on the front side of the dimer interface (Fig. 1B). Interestingly, the dimer interface (Fig. 5A), and the middle band of the front side of the dimer (Fig. 5B), where those five CMT residues are clustered, consistently have a neutral (white) to positively charged (blue) electrostatic surface. In contrast, the rest of the molecule has peculiar and prominent large surfaces that are negatively charged (red) (Fig. 5 B–D). (The positively charged surface where CMT-residues are clustered only partially overlaps with the tRNA interface.) Among the 10 reported CMT-causing mutations, six have a net charge increase (E71G, P234KY, G240R, H418R, D500N, and G526R), whereas none has a charge decrease. This observation suggests that there may be some sort of role for electrostatic interactions in the function of GlyRS that is connected to neurodegeneration and CMT. This role may be linked to, or in addition to, the role of the dimer interface. Although the G526R mutation strengthens the dimer interface (10), it only had a subtle effect on the electrostatic surface potential within the dimer interface (Fig. 5E). However, remarkably, it causes an increase in positive charge density in the central region of the front side of the dimer (Fig. 5F), where many other CMT-associating residues are clustered.

Fig. 5.

Electrostatic surface potential of WT (A–D) and G526R (E and F) GlyRSs. The electrostatic surface is calculated by APBS (15), and rendered in blue and red to illustrate electrostatically positive and negative regions, respectively, in the spectrum ranging from +3 k_BT/e to −3 k_BT/e. (A) The dimer interface on the subunit has an essentially neutral electrostatic distribution, with the tRNA binding side (right side) more positively charged and, therefore, complementary to the negatively charged tRNA backbone. The orientation of the subunit is the same as in Fig. 1B. (B) The front of the dimer has the central band that is mostly positive-charged. Five of the nine resolved CMT-associated residues are on or close to the front surface in the central band. Outside the central band, a large patch of negatively charged surface is located on each side across the dimer. The orientation of the dimer is the same as in Fig. 1A. (C) The opposite side of the subunit dimer interface shown in A. This side is exposed when a dimer is formed. [The orientation is the same as figure 1a of Xie et al. (10).] This side has a strikingly polar charge distribution. In contrast to the positively charged (tRNA binding) face shown as the left side, the rest of the surface is, uniformly, negatively charged, including one side of the anticodon-binding domain. Because insertion III is disordered and, therefore, not included in the calculation of the electrostatic surface potential, the charge distribution on the surface of the catalytic domain can be different when this insertion is included. However, the positively charged surface of the central band in B is not likely to be affected by insertion III. (D) The “back” of the dimer has two negatively charged lobes in the middle of the dimer interface, each being part of the anticodon binding domain. Although the two domains are close to each other in the dimer, they make no contact, possibly because of mutual repulsion. (E) Subunit dimer interface of G526R mutant GlyRS, which is similar to that of the WT shown in A. (F) Front dimer interface of G526R dimer. An increase in positive charges in the center region is apparent, when compared with the WT dimer shown in B.

Having ruled out a general role for aminoacylation (or apparently for protein synthesis) in CMT, the unknown function of GlyRS may be specific to axon development, and is linked to its ability to distribute in nascent, sprouting neurites. As shown in Fig. 4, whereas WT GlyRS distributes into the sprouting neurites of N2a neuroblastoma cells, all of the tested mutant enzymes were defective in their distribution. This result demonstrates a function that is common to all CMT-associated mutant GlyRSs. Thus, if there is a role for the unusual electrostatic pattern seen in Fig. 5, then some part of that electrostatic surface may interact with other partners that, in turn, affect the distribution of GlyRS into neurites and possibly neurons.

Materials and Methods

Immunoprecipitation for Dimer Detection, Analytical Ultracentrifugation, and Aminoacylation Assays.

Those experiments were done as described in ref. 10.

Cell Culture, Differentiation, Staining, and Fluorescence Microscopy.

Mouse neuroblastoma cell line N2a was cultured in Eagle's minimum essential medium, containing 2 mM l-glutamine, Earle's BSS, 1.5 g/liter sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate and 10% FCS. N2a cell differentiation was induced by serum starvation (1% FCS) for 24 h. Cover glasses (0.17–0.25 mm thick with size of 18 mm; Fisher, Tustin, CA) were sterilized and placed in 12-well plates before cells were seeded. Cells were fixed in 2% paraformaldehyde in PBS for 20 min, permeabilized in 0.2% Triton X-100 in PBS for 2 min, rinsed in PBS twice and then blocked in 10% goat serum for 1 h. First, cells were incubated with monoclonal anti-V5 antibodies at a dilution of 1:200 for 1 h, followed by incubation with goat anti-mouse IgG conjugated with Texas Red (1:200 dilution) (Invitrogen, Carlsbad, CA) for 1 h. Polyclonal anti-GlyRS antibodies (in serum) were then added into cells at 1:100 dilution for 1 h followed by incubation with goat anti-rabbit IgG conjugated by FITC for 1 h (1:200 dilution) (Vector Laboratories, Burlingame, CA). (All antibodies were diluted in 2% normal goat serum.) Washings with PBS (3X) were done after every incubation with antibodies. Samples were counterstained with SlowFade Gold antifade reagent with DAPI (Invitrogen) and then mounted on slides. Slides were visualized and analyzed under a Bio-Rad (Zeiss, Thornwood, NY) Radiance 2100 Rainbow laser scanning confocal microscope (LSCM) attached to a Nikon (Tokyo, Japan) TE2000-U microscope with infinity corrected optics. Fluorescence intensities were calculated by Adobe Photoshop (Adobe Systems, San Jose, CA).

Abbreviations

CMT

Charcot–Marie–Tooth disease

GlyRS

glycyl-tRNA synthetase

TyrRS

tyrosyl-tRNA synthetase.

Note Added in Proof.

After submission of this paper, Chihara et al. (16) described a mutation in GlyRS in Drosophila melanogaster that affected elaboration and stability of terminal arborization of axons and dendrites. The mutation (P98L) is also located at the dimer interface.