Long-range structural effects of a Charcot-Marie-Tooth disease-causing mutation in human glycyl-tRNA synthetase - PubMed (original) (raw)
Comparative Study
. 2007 Jun 12;104(24):9976-81.
doi: 10.1073/pnas.0703908104. Epub 2007 Jun 1.
Affiliations
- PMID: 17545306
- PMCID: PMC1891255
- DOI: 10.1073/pnas.0703908104
Comparative Study
Long-range structural effects of a Charcot-Marie-Tooth disease-causing mutation in human glycyl-tRNA synthetase
Wei Xie et al. Proc Natl Acad Sci U S A. 2007.
Abstract
Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structures are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located approximately 30 A away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Overall structure of human GlyRS. (A) “Front” view of the asymmetric unit that contains one subunit of the dimeric enzyme. Different domains and insertions of the subunit are colored as indicated. The three conserved sequence motifs (M1, M2, and M3) in the catalytic domain are colored magenta, pink, and orange, respectively. (B) A 90° rotation along the x axis to view the interface between two subunits of the GlyRS dimer. The second subunit is generated by symmetry operation and is not colored. The β8–β9 hairpins are labeled on both subunits to show their interaction on top of the dimer interface.
Fig. 2.
Structural alignment of human and T. thermophilus GlyRS with the secondary structure elements placed on top. Ten CMT-causing mutations are marked with vertical arrows.
Fig. 3.
G526R mutation blocks the AMP binding site and thereby inactivates the enzyme. (A) The electron density of the region of G526 in wild-type GlyRS. (B) Gly-AMP modeled in the active site of wild-type human GlyRS with potential interactions between G526 and the AMP moiety. (C) The G526R mutation site is clearly shown by the electron density in the mutant structure. A chloride ion was fitted into the extra observed electron density. (D) G526R mutation in the active site blocks the AMP binding site. (E) Loss of overall aminoacylation activity by G526R mutation. (F) Inactivation of the glycine-dependent ATP–PPi exchange activity by G526R mutation. (Inset) Active-site titration of the G526R mutant form of GlyRS demonstrates a significant defect in the formation of glycyl-adenylate as compared with the wild-type enzyme.
Scheme 1.
Scheme 2.
Fig. 4.
G526R mutation strengthens dimer interaction. (A) “Back” view of the GlyRS subunit that shows the dimerization interface. This view is related to the “front” view in Fig. 1_A_ by a 180° rotation along the y axis. The three patches that give dimer interactions are colored: patch 1 (F78–T137) in cyan, patch 2 (F224–L242) in green, and patch 3 (L252–E291) in gold. (B) Loose dimerization interface of wild-type GlyRS generated by mapping the surface area of one subunit that is within 7 Å of the other. (C) The same dimerization interface generated for G526R mutant. The extra dimer interface, absent in the wild-type enzyme, lies in the anticodon recognition domain and is ≈30 Å away from the mutation site. (D) Analytical ultracentrifugation experiment showing that more dimers are formed by G526R mutant than by wild-type GlyRS. (Inset) Immunoprecipitation experiment showing that G526R mutant GlyRS pulled down more endogenous GlyRS than did the wild-type GlyRS, presumably by forming dimers.
Fig. 5.
G41R mutation in TyrRS resembles G526R mutation in GlyRS. (A) Active site of human TyrRS bound with substrate analog tyrosinol. (B) CMT-causing mutation G41R would block tyrosine binding in a similar way as G526R in GlyRS blocks binding of the AMP moiety.
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