Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

How do SMA-linked mutations of SMN1 lead to structural/functional deficiency of the SMA protein?

How do SMA-linked mutations of SMN1 lead to structural/functional deficiency of the SMA protein?

  • Wei Li
PLOS
x

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease with dysfunctional α-motor neurons in the anterior horn of the spinal cord. SMA is caused by loss (∼95% of SMA cases) or mutation (∼5% of SMA cases) of the survival motor neuron 1 gene SMN1. As the product of SMN1, SMN is a component of the SMN complex, and is also involved in the biosynthesis of the small nuclear ribonucleoproteins (snRNPs), which play critical roles in pre-mRNA splicing in the pathogenesis of SMA. To investigate how SMA-linked mutations of SMN1 lead to structural/functional deficiency of SMN, a set of computational analysis of SMN-related structures were conducted and are described in this article. Of extraordinary interest, the structural analysis highlights three SMN residues (Asp44, Glu134 and Gln136) with SMA-linked missense mutations, which cause disruptions of electrostatic interactions for Asp44, Glu134 and Gln136, and result in three functionally deficient SMA-linked SMN mutants, Asp44Val, Glu134Lys and Gln136Glu. From the computational analysis, it is also possible that SMN’s Lys45 and Asp36 act as two electrostatic clips at the SMN-Gemin2 complex structure interface.

Introduction

The spinal muscular atrophy-determining genes

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease with α-motor neuron dysfunction and muscular atrophy [1]. SMA is caused by loss (∼95% of SMA cases) or mutation (∼5% of SMA cases) of the survival motor neuron gene 1 SMN1 (telomeric SMN, telSMN or SMN1, GenBank: U18423) [2]. SMN1 exists in the 5q13 region of the human chromosome, a nearly identical survival motor neuron 2 gene SMN2 (centromeric SMN, cenSMN or SMN2, GenBank: NM_022875) also exists in the 5q13 region [2]. The two genes have been extensively characterized and reviewed in detail [18].

The survival motor neuron protein in the pathogenesis of SMA

The survival motor neuron (SMN) protein is the product of the SMA-determining survival motor neuron gene SMN1, therefore, it is also called the SMA protein. The 38-kD SMN protein locates in the cytoplasm and the nucleus of all cells [911]. In the nucleus, SMN (formerly termed Gemin1) is found in dot-like nuclear structures called gems [2, 12].

In the molecular pathogenesis of SMA, of particular interest is an exon 7-skipping splicing defect identified in the pre-mRNA editing of the SMN2 gene [4]. Due to this splicing defect, SMN2 predominantly produces exon 7–skipped transcripts, which encodes a truncated isoform of the SMN protein (SMNΔ7 or SMN2 with 282 residues, in comparison with the full-length SMN protein with 294 residues (SMN1 or FL-SMN).

In pre-mRNA editing, spliceosome is the major functional unit, spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery [1316]. Here, in SMA, the SMN protein with an A111G mutation (SMNA111G) is capable of snRNP assembly, and can rescue SMA mice that lack Smn and contain either one or two copies of SMN2. This correction of SMA was directly linked to the correction of snRNA levels and snRNP assembly activity in the spinal cord, which supports snRNP assembly as the critical function affected in SMA and suggests that the levels of snRNPs are critical to motor neurons [17].

In the development of SMA, the SMN protein plays an important role in pre-mRNA processing, because the biogenesis of spliceosomal snRNPs is promoted by the SMN complex [14, 18, 19], within which SMN forms oligomers and directly interacts via its N-terminus with Gemin2 and via its Tudor domain with spliceosomal (Sm) proteins [13, 20, 21]. Moreover, the SMA disease severity was also found to correlate with the oligomerization of SMN, which is required before the formation of SMN complexes [22]. A gel filtration investigation showed that most of the refolded SMN was octameric (∼400 kD) or larger [22, 23], the exact number of SMN monomers in an SMN complex is not known yet, nor is it known whether the oligomerization is dependent on the concentration of the SMN protein [6].

To investigate how SMA-linked mutations of SMN1 lead to structural/functional deficiency of SMN, a set of computational analysis of SMN-related structures were conducted and are described in detail in the following Methods section.

Methods

SMA-linked SMN1 mutations (including missense, nonsense, insertion and deletion mutations) exist throughout the SMN protein (Fig 1), and are inextricably linked to the still enigmatic molecular pathogenesis of SMA [6, 24, 25].

thumbnail
Fig 1. The amino acid sequence and point mutation map of SMN.

The amino acid sequence of SMN with SMA-linked SMN1 point mutations highlighted with white texts and black backgrounds.

https://doi.org/10.1371/journal.pone.0178519.g001

Experimentally determined SMN-related structures

For the computational analysis, the SMN-related structures (Table 1) are retrieved from the wwPDB database [30] with two search parameters (Text Search for: survival motor neuron protein and Molecule: Survival motor neuron protein) available as of May 7, 2017.

thumbnail
Table 1. SMN-related structures from the wwPDB database [30].

In this table, TBP (for 4qq6) means to be published, NA (for 4nl6 and 4nl7) means that the structure was from the wrong sample and withdrawn at the request of the author, NumMDL represents the number of structure models in the PDB format file.

https://doi.org/10.1371/journal.pone.0178519.t001

Computational analysis of the SMN-related structures

For the SMN-related structures listed in Table 1, a set of computational analysis was conducted, including salt bridge, hydrogen bond and solvent accessible surface area (SASA) analysis.

First, a simple VMD script was used to split each SMN-related NMR ensemble (multiple-frame PDB file) into a set of single-frame PDB format files. The tcl script is provided as a supplementary file (S3 File).

The salt bridge analysis was conducted with an in-house python script only for titrateable residues (Asp, Glu, Lys, Arg and His), 4.0 Å was used as the cutoff distance for the two oppositely charged groups [31]. The python script is provided as a supplementary file (S4 File).

The hydrogen bond analysis was also conducted for only side chain nuclei with an in-house python script, and employed two geometric criteria: (a) a cutoff value of the angle formed by acceptor (A), donor (D) and hydrogen (H) (∠ADH) of 30°; (b) a cutoff value of donor-acceptor distance at 3.0 Å. That is, a hydrogen bond is only considered to be formed if ∠ADH is not larger than 30° and the donor-acceptor distance is not larger than 3.0 Å [32]. The python script is provided as a supplementary file (S5 File).

The SASA values were calculated by the DSSP program [32, 33] for all residues in the structurally determined region of the SMN-related structures (Table 1), the intrinsic SASA values used here are the standard SASA values used by NACCESS [34].

Results

The critical function that is affected in SMA is snRNP assembly, and it is well known that the snRNP biogenesis pathway involves four steps: (A) SMN oligomerization [22], (B) SMN interaction with Gemin proteins [27], (C) SMN binding to the Sm proteins of snRNPs, which are key constituents of spliceosomes [15], (D) SMN-coilin interaction in Cajal bodies [5], the nuclear organelles involved in the maturation of spliceosomal snRNPs.

In the computational analysis here, a residue is defined as potentially important if its side chain is found to be involved in a salt bridge or a hydrogen bond, or its SASA is smaller than 30% of its standard SASA value. These potentially important SMN residues are listed in the first two columns of Table A in S2 File. Among them, only seven residues (Asp44, Trp92, Trp102, Ala111, Ile116, Glu134 and Gln136) were found to be involved in SMA-linked point mutations of SMN1, as highlighted with white texts and black backgrounds in Table A in S2 File.

Asp44 in the Gemin2-binding domain of SMN

Asp44 is in the exon 2a of SMN1 (the Gemin2-binding domain), and involved in an SMA-linked Asp44Val (D44V) missense mutation [35], which involves a substitution of Asp44’s charged side chain by Val44’s hydrophobic side chain.

Of extraordinary functional significance is that SMN’s Gemin2 binding activity is totally suppressed by the D44V mutation in SMN1 [27]. Moreover, the D44V SMN mutant (SMND44V)’s snRNP assembly activity is lower than that of the wild-type SMN (FL-SMN or SMN1) [36].

In the snRNP assembly, one important participant is the SMN-Gemin2 complex, whose core structure was determined by solution-state nuclear magnetic resonance (NMR) spectroscopy (PDB ID: 2leh, number of structure models in the NMR ensemble is 32, i.e., NumMDL = 32) [27].

In the computational analysis of the SMN-Gemin2 complex (PDB ID: 2leh), three salt bridges were identified between the buried side chains (Table 2) of two charged residues, i.e., SMN’s Asp44 (B44Asp) and Gemin2’s Arg213 (A213Arg), the distance between the geometric centers of two oppositely charged groups was 3.50 ± 0.23 Å.

thumbnail
Table 2. SASA values of SMN’s Asp44 and Gemin2’s Arg213 (PDB ID: 2leh) [21].

In this table, SASA-Mean, SASA-Std, SASA-Intrinsic and SASA-Ratio represent for SMN’s Asp44 and Gemin2’s Arg213 the average SASA value, one standard deviation from the average SASA value, the intrinsic SASA value [34], and the ratio of SASA-Mean divided by SASA-Intrinsic, respectively.

https://doi.org/10.1371/journal.pone.0178519.t002

Specifically, for the side chain charged groups of SMN’s Asp44 and Gemin2’s Arg213, the distance distribution for all 32 NMR structure models (PDB ID: 2leh) is 8.33 ± 2.55 Å for the Oδ2-Nη2 nucleus pair, and 8.71 ± 1.82 Å for the Oδ2-Nη1 nucleus pair, and 8.08 ± 3.02 Å for the Oδ1-Nη2 nucleus pair, and 8.59 ± 2.27 Å for the Oδ1-Nη1 nucleus pair. These distance distributions argue against the existence of the Asp44-Arg213 salt bride, although three were computationally identified in the NMR ensemble (PDB ID: 2leh, NumMDL = 32).

Yet, in an subsequent analysis of the experimental NMR restraints deposited in the BMRB database (PDB ID: 2leh, BMRB ID: 17711) [21], a set of chemical shift assignments were found to be missing for a group of crucial side chain nuclei, including Cγ of SMN’s Asp44 and Cζ, all side chain nitrogen nuclei and all nitrogen-bonded side chain hydrogen nuclei of Gemin2’s Arg213. The missing assignments of those chemical shifts increases the degree of geometric freedom for the side chains in the experimental structure determation, and result in inadequate accuracy of the geometric definition of the Asp44’s and Arg213’s side chains in the NMR ensemble (PDB ID: 2leh).

Taken together, it is conceivable that the buried side chains of SMN’s Asp44 and Gemin2’s Arg213 form a salt bridge, which constitutes a favourable electrostatic energy contribution to the SMN-Gemin2 complex structural stability [27], and highlights the functionally indispensable roles of the two residues’ charged side chains, considering the experimental observation that the SMN-Gemin2 binding is abrogated by the D44V mutation [27], resulting in a functionally deficient SMA-linked D44V SMN mutant (SMND44V).

In a comparison, Lys41 is a positively charged residue and also a neighbouring residue of Asp44. Functionally different to the SMA-linked D44V mutation, a Lys41Ala (K41A) mutation (not SMA-linked) does not affect SMN-Gemin2 binding [27]. This experimental observation is consistent with the salt bridge analysis of the SMN-Gemin2 complex (PDB ID: 2leh, NumMDL = 32), where 15 salt bridges were identified between the side chains of SMN’s Asp35 and Lys41, with the distance between two oppositely charged groups being 3.37 ± 0.39 Å, and of particular relevance, in the SMN-Gemin2 complex (PDB ID: 2leh), SMN’s Lys41 only forms side bridges with SMN’s Asp35, i.e., the 15 Lys41-Asp35 salt bridges are invariably intramolecular, i.e., within the apo SMN protein, instead of intermolecular, i.e., at the SMN-Gemin2 complex structure interface.

SMN’s Lys45 and Asp36 at the SMN-Gemin2 interface

At the SMN–Gemin2 complex structure interface (PDB ID: 2leh), SMN’s Asp36 is directed towards the Gemin2 interface, allowing it to interact with Gemin2’s His120 [21]. In the computational analysis of the SMN-Gemin2 complex [27], four salt bridges were identified between the side chains of SMN’s Asp36 (B36Asp) and those of Gemin2’s His120 (A120His) and His123 (A123His), and four hydrogen bonds were found to be formed between SMN’s Lys45 (B45Lys) and Gemin2’s Gln109 (A109Gln), and between B45Lys and Gemin2’s Gln105 (A105Gln), and between SMN’s Asp36 (B36Asp) and Gemin’s Trp124 (A124Trp) in the NMR ensemble of the SMN-Gemin2 complex (PDB ID: 2leh) (Fig 2) [21].

thumbnail
Fig 2. Asp36 and Lys45 at the binding interface of the SMN-Gemin2 complex.

In this figure, salt bridges are colored differently as sticks on the left side, and hydrogen bonds as red sticks on the right side, each residue is identified with the chain ID (A for Gemin2 and B for SMN), residue number and three-letter residue code, such as B45Lys. The details of the salt bridges and hydrogen bonds are included in Tables 3 and 4.

https://doi.org/10.1371/journal.pone.0178519.g002

thumbnail
Table 3. Two salt bridge pairs formed between A123His and B36Asp and B36Asp and A120His in the fifteenth structure model of the NMR ensemble (PDB ID: 2leh) of the SMN-Gemin2 complex [21].

In the first column, SB represents salt bridge, Inter-atomic distance includes both the distance between the two oppositely charged groups and the distribution of the distances between the two oppositely charged groups for the 32 NMR structure models in the SMN-Gemin2 complex. Color represents the color of the dash lines used to represent the salt bridges in Fig 2 in the main file. In the residue naming scheme here, such as B36Asp, B represents the chain ID used in the PDB file (PDB ID: 2leh), 36 represents the residue number, Asp represents the three-letter code for the amino acid.

https://doi.org/10.1371/journal.pone.0178519.t003

thumbnail
Table 4. Four hydrogen bonds formed between B45Lys and A109Gln and B45Lys and A105Gln of the NMR ensemble of the SMN-Gemin2 complex (PDB ID: 2leh) [21].

Apart from those mentioned in this table, no more hydrogen bond or salt bridge was found for B45Lys. In the first row, HB represents hydrogen bond, DA and HA represent the distance between donor and acceptor and hydrogen and acceptor, respectively. HB1, HB2, HB3 and HB4 represent hydrogen bonds that were computationally identified from the eighth, the eighth, the nineteenth and the eleventh structure models of the SMN-Gemin2 complex (PDB ID: 2leh, NumMDL = 32). The unshaded rows represent the values of DA, HA and ∠ADH for the hydrogen bonds. The shaded rows represent the the distributions of DA, HA and ∠ADH for the 32 NMR structure models in the SMN-Gemin2 complex, i.e., the mean ± one standard deviation of the distances or the ∠ADH angles for the hydrogen bonds. The residue naming scheme is the same as that used in Table 3.

https://doi.org/10.1371/journal.pone.0178519.t004

In a visual inspection of Fig 2, the side chains of SMN’s Lys45 and Asp36 are buried in the groove of the SMN-Gemin2 interface, as also supported by the quantitative analysis in Table 5 of the SASA values of SMN’s Lys45 and Asp36 (PDB ID: 2leh) [21].

thumbnail
Table 5. SASA values of SMN’s Lys45 and Asp36 (PDB ID: 2leh) [21].

In this table, SASA-Mean, SASA-Std, SASA-Intrinsic and SASA-Ratio represent for SMN’s Lys45 and Asp36 the average SASA value, one standard deviation from the average SASA value, the intrinsic SASA value [34], and the ratio of SASA-Mean divided by SASA-Intrinsic, respectively.

https://doi.org/10.1371/journal.pone.0178519.t005

For the salt bridges and the hydrogen bonds described in Fig 2 and Tables 3 and 4, only one/two NMR structure model(s) was/were identified for whole NMR ensemble (PDB ID: 2leh, Num = 32) [21], which presents a strong evidence against the existence of the computationally identified electrostatic interactions described in Fig 2.

Yet, in an analysis of the experimental NMR restraints deposited in BMRB database (PDB ID: 2leh, BMRB ID: 17711) [21], a set of chemical shift assignments were found to be missing for a group of crucial side chain nuclei, including Cγ of SMN’s Asp36 and all four nuclei of the side chain NH3 group of SMN’s Lys45, the two Cδ nuclei of Gemin2’s Gln109 and Gln105, and Cγ, Hδ1 and Hε2 of Gemin2’s His120, and Cγ, Cδ2, Hδ1 and Hε2 of Gemin2’s His123, Cζ3, Cδ2, Hε3, Hζ3, Cγ, Cε2 and Cε3 of Gemin2’s Trp124. The missing assignments of those chemical shifts increases the degree of geometric freedom for the side chains in the experimental structure determation, and result in inadequate accuracy of the geometric definition of the two residues’s side chains which are instrumental in the electrostatic interactions shown in Fig 2.

Taken together, it is reasonable to not rule out the possibility that the deeply buried side chains of SMN’s Lys45 and Asp36 act as two electrostatic clips at the SMN-Gemin2 interface via interactions with Gemin2’s Gln105, Gln109, His120, His123 and Trp124, and that, similar to SMN’s Asp44 and Gemin2’s Arg213, the two SMN residues (Lys45 and Asp36) play stabilizing roles in the SMN-Gemin2 complex structure formation, suggesting that it could be merely a matter of chance in the evolution of the SMN1 gene that no SMA-linked mutations of SMN’s Lys45 and Asp36 have been identified in SMA patients yet, in light of the experimentally observed SMA-linked missense mutation of D44V in the SMN1 gene [35].

Four deeply buried hydrophobic SMN residues

Trp92, Trp102, Ala111 and Ile116 are four hydrophobic SMN residues with SMA-linked mutations in the SMN Tudor domain. With SASA values smaller than 30% of their standard values, Trp92 (12±2%), Trp102 (10±3%), Ala111 (<1%) and Ile116 (14±0.5%) are defined here as deeply buried hydrophobic SMN residues. Furthermore, for the four residues, no salt bridge or hydrogen bond was identified in the computational analysis of the SMN-related structures.

The W92S substitution reduced the binding of the Tudor domain to Sm-B protein by ∼80% [37]. Moreover, in a protein expression study, the mutant W92S SMN protein was expressed at lower levels than the wild-type SMN protein, but no difference was observed in the transcript levels, suggesting the instability of the SMN protein due to the W92S mutation [38].

Trp102 is involved in a nonsense SMA-linked mutation (Trp102X), a premature termination mutations in SMN1 exon 3, which was identified in two SMA patients with a relatively mild phenotype who had two copies of SMN2 [39, 40]. More functional study is required for this SMA-linked mutation.

Another hydrophobic residue in the SMN Tudor domain, Ala111 is almost completely buried in the SMN Tudor domain (with an SASA value of 0.3 ± 0.48 Å2 compared with its standard SASA value at 107.9 Å2). The Ala111Gly (A111G) mutation [17, 41] reduces, but does not totally suppress, the binding of SMN to Sm proteins, confirming Tudor domain‘s role in SMN‘s binding to Sm proteins [15, 16].

For the A111G mutation, the net structural impact is the removal of the hydrophobic side chain of Ala111 (similar to the A2G mutation). Moreover, as discussed above, the SMNA111G mutant is still capable of snRNP assembly and associating with full-length SMN to form an oligomer in vitro [17]. Collectively, it is conceivable that A111’s deeply buried hydrophobic side chain points towards the interior of the SMN Tudor domain structure and provides a favourable energy contribution to the Tudor domain stability, this intra-domain impact might help explain the reduced (but not abrogated) binding of SMN to Sm proteins.

Ile116 is involved in an SMA-linked Ile116Phe (I116F) missense mutation which was first reported in Spanish patients [24, 25]. The Sm core assembly activity of I116F SMN mutant was found to be significantly lower than that of SMN1 [42].

Taken together, the four SMA-linked mutations highlight the potential significance of the deeply buried hydrophobic side chains of Trp92, Trp102, Ala111 and Ile116 in the SMN Tudor domain. More comprehensive structural and functional research is required to characterize the functional roles of these residues in SMN and also the development of SMA.

Glu134 and Gln136 in the SMN Tudor domain

Glu134 and Gln136 are involved in two SMA-linked mutations in the SMN Tudor domain, i.e., Glu134Lys (E134K) and Gln136Glu (Q136E), respectively.

Both SMN’s Glu134 and Gln136 are involved in hydrogen bonds with SMN’s Tyr127, as revealed by both the structures of apo SMN Tudor domain [26, 43] and the SMN Tudor domain bound to symmetric and asymmetric dimethylated arginine (sDMA and aDMA) [28], and also by the computational analysis here, where Gln136 was found to form 14 hydrogen bonds with Tyr127 (PDB ID: 4a4g, NumMDL = 20), and Glu134 was found to form 20 (PDB ID: 4a4e, NumMDL = 20) and 19 (PDB ID: 4a4g, NumMDL = 20) hydrogen bonds with Tyr127. In addition, Glu134 also formed 18 (PDB ID: 4a4e, NumMDL = 20) and 12 (PDB ID: 4a4g, NumMDL = 20) hydrogen bonds with SMN’s Ser103.

In the NMR structure of SMN Tudor domain bound to sDMA [28], the E134K mutation reduced binding affinity for sDMA by an order of magnitude. In addition, the Sm core assembly activity of both SMN mutants (SMNE134K and SMNQ136E) were found to be significantly lower than the wild-type SMN [42].

Regardless of whether new salt bridges formed or not due to the two SMA-linked substitutions E134K and Q136E), one approach to understand the above-described functional deficiency of SMN is to structurally examine the two mutations’ consequence(s) at residue positions 134 and 136 in SMN. In the computational structural analysis, local electrostatic interaction disruptions in SMN Tudor domain do arise due to the disruptions of the Glu134- and Gln136-involved hydrogen bond interactions.

For instance, Glu134 provides its Oε1 or Oε2 as the hydrogen acceptor in the hydrogen bonds, where the two oxygens carry partial negative charges, and the hydrogen bond donor carries partial positive charge. An E134K substitution removes the negatively charged side chain group of Glu134, and installs a positively charged lysine side chain instead. This reversal in the side chain electric charge causes a shift of electrostatic attraction (the hydrogen bond interactions between Glu134 and Tyr127, and between Glu134 and Ser103) to electrostatic repulsion between positively charged Lys134 side chain and the hydrogen bond donor which carries partial positive charge, resulting in local electrostatic interaction disruption in the SMN Tudor domain structure.

Similarly, Gln136 provides its side chain NH2 group as the hydrogen donor group in the hydrogen bonds, where the hydrogen carries partial positive charge due to its low electronegativity compared with the directly bonded nitrogen, and the hydrogen bond acceptor carries partial negative charge. A Q136E substitution removes the neutral side chain group of Gln136, along with the hydrogen with partial positive charge in the side chain NH2 group of Gln136, and installs a negatively charged glutamate side chain instead. This reversal in the side chain electric charge causes a shift of electrostatic attraction (the hydrogen bond interaction between Gln136 and Tyr127) to electrostatic repulsion between negatively charged Glu136 side chain and the hydrogen bond acceptor which carries partial negative charge, resulting in local electrostatic interaction disruption in the SMN Tudor domain structure.

Overall, the structural consequences of the E134K and the Q136E mutations consist qualitatively of two reversals in the signs of the electrostatic interactions (from attractions to repulsions) involving the two SMN residues at positions 134 and 136. As a result, the two SMA-linked mutations in the Tudor domain constitute two local electrostatically destabilizing sites, contributing to the structural instability of the SMN Tudor domain (the essential part of SMN for the Sm protein-binding), which can help explain the reduced Sm core assembly activity of the two SMA-linked SMNE134K and SMNQ136E mutants.

Conclusion

To characterize the relationship between SMA-linked mutations of SMN1 and SMN’s structure and function, a set of computational analysis of SMN-related structures was conducted and described above. With the structural analysis, this article highlights three residues of SMN (Asp44, Glu134 and Gln136), and the electrostatic basis (Fig 3) of how the SMA-linked missense mutations of the three residues cause structural/functional deficiency of SMN, and also a possibility of SMN’s Lys45 and Asp36 acting as two electrostatically stabilizing clips at the SMN-Gemin2 complex structure interface.

thumbnail
Fig 3. Disruptions of local electrostatic interactions by three SMA-linked missense mutations.

In this figure, the electrostatic interaction disruptions for D44V, E134K and Q136E are illustrated in the upper, the middle and the lower panels, respectively. In the three panels, the negative (partial, for hydrogen bond acceptor group) charge is represented with a red circle with a minus sign inside, the positive (partial, for hydrogen bond donor group) charge is represented with a blue circle with a plus sign inside, the salt bridge/the hydrogen bond is represented with a set of green squares, representing electrostatic attraction. For the upper panel, the impact of the mutation is represented with the disappearance of the green squares on the right side, representing the disappearance of the Asp44-Arg213 salt bridge. For the middle and lower panels, the impacts of the mutations are represented with a set of red squares, representing electrostatic repulsion.

https://doi.org/10.1371/journal.pone.0178519.g003

In addition, the structural analysis here also suggested potential functional significance of four deeply buried hydrophobic residues (Trp92, Trp102, Ala111 and Ile116) with SMA-linked point mutations in the SMN Tudor domain.

Discussion

Since the SMN protein (also called the SMA protein) has an established role in snRNP biogenesis and the development of SMA, SMA Patient-derived SMN mutations offer a valuable resource to help characterize the function of SMN. While the functional impact was studied for certain SMA-linked mutations, due to the lack of experimentally determined three-dimensional structure, a structural analysis is not possible yet for those SMN residues with SMA-linked mutations.

For example, in zebra-fish, a Gly264Asp (G264D) missense mutation is linked to presynaptic neuromuscular junction defect via the synaptic vesicle protein SV2 [44]. Interestingly, the G264 of zebra-fish aligns with G279 of human in the amino acid sequence alignment [44]. Moreover, G279 locates in the truncated part of SMNΔ7 compared with FL-SMN, and two SMA-linked missense mutations (G279C and G279V) were also identified for this glycine [3, 42], highlighting the functional significance of Gly279 (and also the truncated C-terminal part) of FL-SMN.

As of May 7, 2017, the wwPDB website [30] produced nine experimentally determined SMN-related structures (Table 1). In terms of amino acid sequence, those SMN-related structures are only SMN fragments, ranging from Gly26 to Lys51, and from Asn84 to Glu147 (S1 File). In between, there is still a structurally not-determined-yet region consisting of 204 SMN residues, calling for continued structural and functional characterization of the SMA protein.

Supporting information

S1 File. Results of the computational analysis of SMN-related NMR structures.

This supplementary file (S1_File.pdf) provides the results of the computational analysis of salt bridge, hydrogen bond and solvent accessible surface area (SASA) for four SMN-related structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy.

https://doi.org/10.1371/journal.pone.0178519.s001

(PDF)

S2 File. Interpretation and visualization of the results of the computational analysis of SMN-related structures.

This supplementary file (S2_File.pdf) presents four supporting figures for the visualization of the salt bridge and the hydrogen bond analysis, a table of computationally identified SMN residues aligned against experimentally identified SMA-linked SMN mutations, and a set of tables describing the chemical shift assignment analysis for the SMN-related SMN structures.

https://doi.org/10.1371/journal.pone.0178519.s002

(PDF)

S3 File. A tcl script for the splitting of SMN-related NMR ensemble into single-frame PDB files.

This tcl script (S3 File) is used in VMD to split NMR ensembles.

https://doi.org/10.1371/journal.pone.0178519.s003

(TCL)

S4 File. A python script for structure-based computational salt bridge analysis.

This supplementary file (S4_File.py) presents a python script for structure-based computational salt bridge analysis. The salt bridge analysis was conducted only for titrateable residues (Asp, Glu, Lys, Arg and His), 4.0 Å was used as the cutoff distance for the two oppositely charged groups.

https://doi.org/10.1371/journal.pone.0178519.s004

(PY)

S5 File. A python script for structure-based computational hydrogen bond analysis.

This supplementary file (S5_File.py) presents a python script for structure-based computational hydrogen bond analysis. The hydrogen bond analysis employs two geometric criteria: (a) a cutoff value of the angle formed by acceptor (A), donor (D) and hydrogen (H) (∠ADH) of 30°; (b) a cutoff value of donor-acceptor distance at 3.0 Å.

https://doi.org/10.1371/journal.pone.0178519.s005

(PY)

Author Contributions

  1. Conceptualization: WL.
  2. Data curation: WL.
  3. Investigation: WL.
  4. Methodology: WL.
  5. Software: WL.
  6. Visualization: WL.
  7. Writing – original draft: WL.
  8. Writing – review & editing: WL.

References

  1. 1. Burghes AHM. When Is a Deletion Not a Deletion? When It Is Converted. American Journal of Human Genetics. 1997;61(1):9–15. pmid:9245977
  2. 2. Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80(1):155–165. pmid:7813012
  3. 3. Lorson CL, Strasswimmer J, Yao JM, Baleja JD, Hahnen E, Wirth B, et al. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nature Genetics. 1998;19(1):63–6. pmid:9590291
  4. 4. Hua Y, Vickers TA, Okunola HL, Bennett CF, Krainer AR. Antisense Masking of an hnRNP A1/A2 Intronic Splicing Silencer Corrects SMN2 Splicing in Transgenic Mice. American Journal of Human Genetics. 2008;82(4):834–848. pmid:18371932
  5. 5. Tapia O, Lafarga V, Bengoechea R, Palanca A, Lafarga M, Berciano MT. The SMN Tudor SIM-like domain is key to SmD1 and coilin interactions and to Cajal body biogenesis. Journal of Cell Science. 2014;127(5):939–946. pmid:24413165
  6. 6. Burghes AH, Beattie CE. Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nature Reviews Neuroscience. 2009;10(8):597–609. pmid:19584893
  7. 7. Hua Y, Sahashi K, Hung G, Rigo F, Passini MA, Bennett CF, et al. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes & Development. 2010;24(15):1634–6144.
  8. 8. Hua Y, Sahashi K, Rigo F, Hung G, Horev G, Bennett CF, et al. Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature. 2011;478(7367):123–216. pmid:21979052
  9. 9. Liu Q, Dreyfuss G. A novel nuclear structure containing the survival of motor neurons protein. Embo Journal. 1996;15(14):3555–3565. pmid:8670859
  10. 10. Carvalho T, Fcalapez Almeida, A , Lafarga M, Berciano M, Carmo FM. The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body. Journal of Cell Biology. 1999;147(4):715–727. pmid:10562276
  11. 11. Young PJ, Le TT, Dunckley M, Nguyen TM, Burghes AH, Morris GE. Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN. Experimental Cell Research. 2001;265(2):252–261. pmid:11302690
  12. 12. Young P, Ntlorson Man C, Le T, Androphy E, Burghes A, Morris G. The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding. Human Molecular Genetics. 2000;9(19):2869–8277. pmid:11092763
  13. 13. Seng CO, Magee C, Young PJ, Lorson CL, Allen JP. The SMN structure reveals its crucial role in snRNP assembly. Human Molecular Genetics. 2015;24(8):2138–46. pmid:25561692
  14. 14. Grimm C, Chari A, Pelz JP, Kuper J, Kisker C, Diederichs K, et al. Structural basis of assembly chaperone-mediated snRNP formation. Molecular Cell. 2013;49(4):692–703. pmid:23333303
  15. 15. Pellizzoni L, Dreyfuss G. SMN Mutants of Spinal Muscular Atrophy Patients Are Defective in Binding to snRNP Proteins. Proceedings of the National Academy of Sciences. 1999;96(20):11167.
  16. 16. Pellizzoni L. Essential Role for the SMN Complex in the Specificity of snRNP Assembly. Science. 2002;298(5599):1775–1779. pmid:12459587
  17. 17. Workman E, Saieva L, Carrel TL, Crawford TO, Liu D, Lutz C, et al. A SMN missense mutation complements SMN2 restoring snRNPs and rescuing SMA mice. Human Molecular Genetics. 2009;18(12):2215–2229. pmid:19329542
  18. 18. Otter S, Grimmler M, Neuenkirchen N, Chari A, Sickmann A, Fischer U. A comprehensive interaction map of the human survival of motor neuron (SMN) complex. Journal of Biological Chemistry. 2007;282(8):5825–8533. pmid:17178713
  19. 19. Chari A, Golas MM, Klingenhäger M, Neuenkirchen N, Sander B, Englbrecht C, et al. An Assembly Chaperone Collaborates with the SMN Complex to Generate Spliceosomal SnRNPs. Cell. 2008;135(3):497–509. pmid:18984161
  20. 20. Liu Q, Fischer U, Wang F, Dreyfuss G. The Spinal Muscular Atrophy Disease Gene Product, SMN, and Its Associated Protein {SIP1} Are in a Complex with Spliceosomal snRNP Proteins. Cell. 1997;90(6):1013–1021. pmid:9323129
  21. 21. Sarachan KL, Valentine KG, Gupta K, Moorman VR, Gledhill JM, Bernens M, et al. Solution structure of the core SMN-Gemin2 complex. Biochemical Journal. 2012;445(3):361–370. pmid:22607171
  22. 22. Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proceedings of the National Academy of Sciences. 1999;96(11):6307–3611.
  23. 23. Man NT, Humphrey E, Lam LT, Fuller HR, Lynch TA, Sewry CA, et al. A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy. Neurology. 2008;71(71):1757–1763.
  24. 24. Alías L, Bernal S, Fuentes-Prior P, Barceló MJ, Also E, Martínez-Hernández R, et al. Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene. Human Genetics. 2008;125(1):29–39. pmid:19050931
  25. 25. Cuscó I, Barceló MJ, Del RE, Baiget M, Tizzano EF. Detection of novel mutations in the SMN Tudor domain in type I SMA patients. Neurology. 2004;63(1):146–149. pmid:15249625
  26. 26. Sattler M, Selenko P, Sprangers R, Stier G, Bühler D, Fischer U. SMN Tudor domain structure and its interaction with the Sm proteins. Nature Structural Biology. 2001;8(1):27–31. pmid:11135666
  27. 27. Zhang R, So BR, Li P, Yong J, Glisovic T, Wan L, et al. Structure of a key intermediate of the SMN complex reveals Gemin2’s crucial function in snRNP assembly. Cell. 2011;146(3):384–395. pmid:21816274
  28. 28. Tripsianes K, Madl T, Machyna M, Fessas D, Englbrecht C, Fischer U, et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nature Structural & Molecular Biology. 2011;18(12):1414–1420.
  29. 29. Martin R, Gupta K, Ninan NS, Perry K, Duyne GDV. The Survival Motor Neuron protein forms soluble glycine zipper oligomers. Structure. 2012;20(11):1929–1939. pmid:23022347
  30. 30. Berman H, Henrick K, Nakamura H. Announcing the worldwide Protein Data Bank. Nature Structural Biology. 2003;10(12):980. pmid:14634627
  31. 31. Xiong JP, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, et al. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science. 2002;296:151–155. pmid:11884718
  32. 32. Kabsch W, Sander C. Dictionary of protein secondary structure: Pattern recognition of hydrogenbonded and geometrical features. Biopolymers. 1983;22(12):2577–2637. pmid:6667333
  33. 33. Joosten RP, Beek TAHT, Krieger E, Hekkelman ML, Hooft RWW, Schneider R, et al. A series of PDB related databases for everyday needs. Nucleic Acids Research. 2011;39:411–419.
  34. 34. Hubbard SJ, Thornton JM. Naccess. Computer Program, Department of Biochemistry and Molecular Biology, University College London. 1993;2.
  35. 35. Sun Y, Grimmler M, Schwarzer V, Schoenen F, Fischer U, Wirth B. Molecular and functional analysis of intragenic SMN1 mutations in patients with spinal muscular atrophy. Human Mutation. 2005;25(1):64–71. pmid:15580564
  36. 36. Ogawa C, Usui K, Aoki M, Ito F, Itoh M, Kai C, et al. Gemin2 plays an important role in stabilizing the survival of motor neuron complex. Journal of Biological Chemistry. 2007;282(15):11122–11134. pmid:17308308
  37. 37. Kotani T, Sutomo R, Sasongko TH, Sadewa AH, Gunadi , Minato T, et al. A novel mutation at the N-terminal of SMN Tudor domain inhibits its interaction with target proteins. Journal of Neurology. 2007;254(5):624–630. pmid:17415510
  38. 38. Takarada T, Rochmah MA, Harahap NIF, Shinohara M, Saito T, Saito K, et al. SMA mutations in SMN Tudor and C-terminal domains destabilize the protein. Brain and Development. 2017; pmid:28366534
  39. 39. Prior TW. Spinal muscular atrophy diagnostics. Journal of Child Neurology. 2007;22(8):952–956. pmid:17761649
  40. 40. Sossi V, Giuli A, Vitali T, Tiziano F, Mirabella M, Antonelli A, et al. Premature termination mutations in exon 3 of the SMN1 gene are associated with exon skipping and a relatively mild SMA phenotype. European Journal of Human Genetics Ejhg. 2001;9(2):113–120. pmid:11313744
  41. 41. Monani UR, Pastore MT, Gavrilina TO, Jablonka S, Le TT, Andreassi C, et al. A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy. Journal of Cell Biology. 2003;160(1):41–52. pmid:12515823
  42. 42. Shpargel KB, Gall JG. Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(48):17372–73717. pmid:16301532
  43. 43. Sprangers R, Groves MR, Sinning I, Sattler M. High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues. Journal of Molecular Biology. 2003;327(2):507–520. pmid:12628254
  44. 44. Boon KL, Xiao S, Mcwhorter ML, Donn T, Wolf-Saxon E, Bohnsack MT, et al. Zebrafish survival motor neuron mutants exhibit presynaptic neuromuscular junction defects. Human Molecular Genetics. 2009;18(19):3615–3625. pmid:19592581