Table 1.
Data processing and refinement statistics.
Data in parentheses refer to the highest-resolution shell.
Fig 1.
A. Location of the mutations studied here, mapped onto the crystal structure of wild-type GDAP1 [11] in two different orientations. At the centre of the dimer interface, a disulphide bridge between Cys88 from each protomer links the dimer covalently. B. The mapping of the mutations studied here (red) onto the AlphaFold2 model, to include those not visible in the crystallised construct (R161H, R310Q, R310W). The TM domain is at the bottom right, and the helix preceding it harbours Arg310. C. Open/close conformations involving the long helix ⍺6 have been observed both experimentally (left) and using structure prediction (right). Closed conformations are in blue and open in grey. D. Hydrogen bonding network of residues on the core helices of GDAP1. E. Same view as D, but known sites of CMT mutations have been added in magenta. CMT mutations are clustered on the GDAP1 core helices.
Fig 2.
Structural details for each point mutation studied by X-ray crystallography in this study.
A. Immediate environment of the R120Q mutation. Arg120 participates in a hydrogen bond network between helices α3 and α6. B. Effects of the A247V mutation. Ala247 on helix α7 is a central residue of the GDAP1 hydrophobic core. C. The R282H mutation. Arg282 interacts with the α6-α7 loop, and the mutation causes loss of these interactions. The structure of wild-type GDAP1 is shown in grey in all panels.
Fig 3.
A. SAXS curves for the mutants in the GDAP1Δ303–358 construct. The curves have been displaced along the y axis for clarity. B. Distance distributions for the curves in A. R161H shows a more open conformation than the other variants. C. Top: Dummy atom model of R282H superimposed with a structure based on the collapsed conformation of wild-type GDAP1Δ295–358 [12]. Bottom: Dummy atom model of the R310Q mutant in GDAP1Δ319–358, superimposed on the same structure, indicating additional volume for the C-terminal extension. D. SAXS curves for the mutants in the GDAP1Δ319–358 construct. The dominant monomer peak from SEC was used for R310W and the dimer peak for R310Q. E. Distance distributions for the curves in D. F. Dimensionless Kratky plots for all constructs show similar levels of rigidity and globularity. The cross marks the location of the peak in a perfect globular particle.
Table 2.
Data for wild-type GDAP1 as well as the monomeric mutation Y29EC88E are taken from [11]. The MW estimate corresponds to the Bayesian estimate from PRIMUS.
Fig 4.
Folding and stability of GDAP1 as affected by CMT mutations.
A. SRCD spectra for the mutants in the GDAP1Δ303–358 construct. The spectral shape is most different for R161H, and for R282H, the spectral amplitude is increased, but the shape does not change compared to wild-type GDAP1. B. SRCD spectra for the mutants in GDAP1Δ319–358. C-D. nanoDSF analysis for the mutants in GDAP1Δ303–358 (C) and GDAP1Δ319–358 (D). The curves shown are each average of three independent nanoDSF curves run in parallel. E. Non-reducing SDS-PAGE analysis of all studied variants. Top: variants in GDAP1Δ303–358; wt1Δ302 refers to the wild-type construct. Bottom: variants in GDAP1Δ319–358; wt1Δ319 refers to the wild-type construct. For the longer constructs (panel below), the added segment is most likely a membrane-binding motif, and the mutations may affect membrane interactions; this could explain the difference in electrophoretic mobility between the wild-type and mutant variants. The segment in question is likely to bind to SDS and form a helical structure instead of getting denatured. The uncropped gel images are in S2 Fig.
Table 3.
nanoDSF apparent melting points.
All values are average ± standard deviation from 3 replicates.
Fig 5.
Structural bioinformatics analysis of the crystallised CMT variants.
A. The C⍺ deviation of each mutant vs. wild-type GDAP1 structure. The highest deviations can be found in the residue range 220–230. Shown are only the residues participating in the hydrogen bonding network. B. Mapping the results onto the structure, it becomes evident that the C-terminal end of helix ⍺6 deviates the most from wild-type GDAP1 on average. C. Average predicted ΔΔG effect of GDAP1 mutations at CMT sites, as defined by CUPSAT. D. CUPSAT predictions for R120Q, A247V, and R282H being mutated into all possible amino acids. Note that a negative ΔΔG in CUPSAT means destabilisation, and that A247V is falsely predicted as stabilising. The mutations studied here are marked with spheres. Ala247, red; Arg120, blue; Arg282, green.
Fig 6.
A. Colour scheme for phylogenetic trees. Note that not all organisms seen in the colour legend are present in every picture. This is because the trees are very large and to increase visibility parts of the tree will be collapsed (denoted by a grey circle), to hide underlying branches. B. The first branch of the phylogenetic tree. The tree was rooted at the root sequence. On the right side are eukaryotic GST sequences (mammals, fish, birds, reptiles). The branch includes 48 sequences. The rest of the 398 sequences that are represented are at hierarchically lower levels of the phylogenetic tree. The corresponding branches were collapsed (grey circle). C. In contrast to panel B, the left branch is partially extended. The right-sided branch shows eukaryotic GST sequences, while the left side shows a total of 110 bacterial GST sequences. The remaining sequences are at hierarchically lower levels of the phylogenetic tree. The corresponding branches were collapsed (grey circle). The red line indicates that the time could not be resolved at this level. D. The last hierarchical level of the phylogenetic tree. The tree was rooted at the root sequence. Purple sequences on the right side correspond to bacterial GST sequences. Sequence for eukaryotic GDAP1 and GDAP1L1 are in the left side branches. The grey circle denotes the collapsed branch for the eukaryotic GSTs (see panel B) and the magenta circle denotes the second group of bacterial GST sequences (see panel C).
Fig 7.
A. Entropy plot for GDAP1. The positions of the mutations studied here are shown with dots, and these positions indicate a high level of conservation (low entropy). B. Mapping of entropy onto the GDAP1 crystal structure monomer. Blue indicates high entropy and red low.
Table 4.
Entropy analysis of the GDAP1 subfamily.
Shown are the positions (human GDAP1 reference sequence numbering) with the lowest entropy (S < 0.1).
Table 5.
Entropy analysis for the large data set including the GST dataset.
Shown are residue positions with the lowest entropy (S < 0.2).
Table 6.
KL1 and KL2 are the Kullback-Leibler divergence using the GDAP1 or GST group as reference distribution. S1 and S2 are the sequence entropy within the two groups. GDAP1 and GST show the most common residue within each group.
Fig 8.
Location of Arg310 outside the GST-like core.
Arg310 within the amphipathic helix preceding the transmembrane domain is predicted to make salt bridges back towards the core domain.
Fig 9.
An overview of the ⍺6-⍺7 loop and its surroundings in light of known CMT mutations and conserved positions.
In this stereo view, the ⍺6-⍺7 loop is coloured orange, green shows positions for CMT mutations, yellow positions that are highly conserved, and blue the positions that are both targeted by CMT mutations and highly conserved. Selected residues are labelled for clarity.
Fig 10.
Comparisons of the studied mutations to a canonical GST from S. japonicum.
GDAP1 is in gray and GST in blue. A. Overall view of the GDAP1-GST superposition, with the CMT mutation sites crystallised here highlighted in pink. B. Arg120 (pink) in GDAP1 and the corresponding interaction in GST, made by an Arg from a nearby helix. C. Tight packing of Ala247 (green) in the GDAP1 structure; Ala at this position is highly conserved across the whole GST family. Note Tyr124, which was highlighted in the KL divergence analysis, making direct van der Waals contact with Ala247. D. The interactions of Arg282 (pink) towards the ⍺6-⍺7 loop and Trp238 are conserved in GST.