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. 2016 Apr 20;44(7):3420-31.
doi: 10.1093/nar/gkw082. Epub 2016 Feb 10.

The crystal structure of human GlnRS provides basis for the development of neurological disorders

Jana Ognjenović  1 , Jiang Wu  2 , Doreen Matthies  3 , Ulrich Baxa  3 , Sriram Subramaniam  3 , Jiqiang Ling  4 , Miljan Simonović  5
Affiliations

The crystal structure of human GlnRS provides basis for the development of neurological disorders

Jana Ognjenović et al. Nucleic Acids Res. .

Abstract

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.

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Figures

Figure 1.
Figure 1.
Overall structure and domain organization of the intact human GlnRS. (A) A cartoon representation of two views of the intact structure of human GlnRS. Domains are labeled as in the main text and colored according to scheme in (C). (B) A surface representation diagram of the structure of hGlnRS in the same orientation as in (A). (C) Domain structure with domain names and coloring scheme. The conserved motifs, ‘HIGH’ and ‘VVSKR’, and connecting subdomain are indicated. Agreement between the final model and electron density maps is shown in Supplementary Figure S1.
Figure 2.
Figure 2.
Structural comparison of domains in human, yeast and bacterial GlnRSs. (A,B) The global superimpositioning of NTDs from human and yeast GlnRS, and GatB reveals significant structural divergence in spite of the overall conservation of the domain architecture (see Supplementary Figure S2). (C,D) The superimpositioning of core domains (CATD, CP1 and ABD) in yeast and bacterial GlnRSs onto that in human GlnRS shows that although the overall fold is conserved, there are significant structural differences in the CP1 and anticodon-binding domains (marked by asterisks; see Supplementary Figure S3). (E) Enzymatic results show the loss of orthogonality between human and E. coli GlnRS and tRNAGln pairs.
Figure 3.
Figure 3.
The pathological mutations, G45V and Y57H, mildly affect the structure and conformation of NTD in human GlnRS. (A) Four mutations implicated in the development of neurological disorder are mapped onto the structure of hGlnRS. Two mutations, G45V and Y57H, are located in NTD, R403W is in CP1 and R515W is in CATD. The pairs of mutations found in different patients are colored blue (G45V and R403W) and yellow (Y57H and R515W). (B) The crystal structure of G45V is similar to that of the WT hGlnRS. A minor structural rearrangement is present in the loop connecting helices α4 and α5 in NTD. (C) The superimposition of Y57H onto hGlnRS reveals that NTD rotates ≈3° toward the CATD in the mutant structure. A close-up view of structural differences in NTDs is shown in the box in (B), and (C).
Figure 4.
Figure 4.
Effect of pathological mutations on the aggregation status and stability of GlnRS. (A) The melting curve of the WT hGlnRS (red line) exhibits a single peak with Tm of +47.5°C, whereas curves of G45V (green line) and Y57H (blue line) have two peaks with distinct minima at ≈+46°C and ≈+52°C. R403W (solid black line) and R515W (dashed line) exhibit a constant decrease in signal intensity without prominent peaks, suggesting formation of aggregates or other complexes. (B) Elution profiles of WT (red line), G45V (green line), Y57H (blue line), R403W (solid black line) and R515W GlnRS (dashed line) from the size-exclusion chromatography (SEC) column. WT, G45V and Y57H form predominantly monomers in solution (retention time ≈95 min), whereas R403W and R515W primarily form larger species that elute ≈60 min. Peaks eluting ≈110 min contain impurities.
Figure 5.
Figure 5.
R403W and R515W mutants are bound to GroEL when recombinantly expressed in E. coli suggesting that they are misfolded. (A) SDS-PAGE (left panel) of purified GlnRS samples (R515W, R403W, G45V, Y57H and WT) reveals that only R->W mutants contain an additional protein band that runs at ≈60 kDa. The western blot with the anti-GlnRS antibodies (middle panel) demonstrates that all samples contain human GlnRS, whereas the same analysis with the anti-GroEL antibodies (right panel) shows that the ‘aggregate’ peaks of R->W mutants contain GroEL as well. (B) The native PAGE and subsequent western blots with anti-GlnRS (middle panel) and anti-GroEL (right panel) antibodies shows that WT, Y57H and G45V run as monomeric proteins (apparent molecular mass between 66 and 120 kDa), whereas R403W and R515W stably associate with GroEL and run as a single species of molecular mass above 720 kDa.
Figure 6.
Figure 6.
Negative-stain electron microscopy of hGlnRS R403W and R515W mutants. (A,B) Representative images of R403W mutant at different magnifications revealing a dominant population of GroEL particles. Three zoomed in insets are shown on the right displaying different views of the chaperonin. (C,D) Representative images of R515W mutant at different magnifications revealing a dominant population of GroEL particles. Three zoomed in insets are shown on the right displaying different views of the chaperonin. Size bars designate 200 nm in panels A and C, and 100 nm in panels B and D.
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