Dispersed disease-causing neomorphic mutations on a single protein promote the same localized conformational opening

Abstract

The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an axonal form of Charcot-Marie-Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator. Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.

Fig. 1.

Distribution of CMT-causing mutations on GlyRS. (A) All 13 CMT-causing mutations mapped onto the domain structure of GlyRS. The three sequence motifs that are characteristic of the catalytic domain of class II tRNA synthetases are noted as 1, 2, 3, and the three insertions to the catalytic domain as I, II, III. (B) Dimer interface location of two newly identified CMT-associated residues C157 and P244. (C) Close-up view of the location of C157 and P244.

Fig. 2.

Changes in deuterium incorporation resulting from CMT-causing mutations or deletion of the WHEP domain. The results are mapped on the primary sequence of GlyRS, with CMT mutation-associated residues highlighted in purple. The percent difference of deuterium incorporation is calculated from the hydrogen-deuterium exchange after 1 h for each mutant relative to WT GlyRS. The uncovered areas for each mutant may result from either lack of sequence coverage for either the mutant or WT GlyRS, or lack of common peptides for direct comparison. (The sequence coverage for WT, L129P, G240R, G526R, S581L, G598A, and ΔWHEP GlyRS themselves were 96%, 87%, 95%, 89%, 98%, 99%, and 96%, respectively.) The eight consensus opened-up areas (hot spots) are labeled. In general, the hot spots are well-covered in at least four of the five CMT mutants we tested, and each mutant peptide within the hot spots has greater than 5% increase in HDX relative to the WT protein. The hot spot areas might slightly increase, if the sequence coverage improves.

Fig. 3.

Changes in deuterium incorporation mapped onto the crystal structure of GlyRS. (A) Mapping of changes in deuterium incorporation caused by different CMT mutations or deletion of the WHEP domain. The monomeric structure is oriented to view the dimerization interface. The color coding is the same as in Fig. 2. (B) Mapping of the consensus areas (or hot spots) that are opened up by all 5 tested CMT-causing mutations. The hot spots are colored in gold and CMT mutation-associated residues in purple. Depicted on the left is a dimeric view of the GlyRS structure.

Fig. 4.

SAXS analysis confirms the structure opening of G526R GlyRS and reveals the conformational change of the WHEP domain. (A) Solution scattering data of WT and G526R GlyRS. Small-angle X-ray scattering curves are overlapped with theoretical scattering profiles calculated from ab initio models (black line). The inset shows Guinier plots at the low-angle region (S ∗ R_g < 1.3). (B) Distance distribution P(R) functions of WT and G526R GlyRS. P(R) curves were calculated from SAXS data shown in (A). The main differences between WT and G526R GlyRS are indicated by arrows. (C) SAXS-based ab initio modeling of WT (purple) and G526R (yellow) GlyRS. The crystal structure of the dimeric WT GlyRS was manually docked into the SAXS-based molecular envelope, leaving two extra densities located on each side of the dimer near the N-terminus of the GlyRS structure. Therefore, the extra densities most likely correspond to the disordered WHEP domain in the crystal structure. The extra densities are fit with a model of the WHEP domain from HisRS (PDB∶1X59) and differ between WT and G526R GlyRS, suggesting a conformational change of the WHEP domain induced by the G526R mutation.

Fig. 5.

Illustration of the same conformational opening of GlyRS induced by different CMT-causing mutations and of the generation of a common neomorphic surface.