Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis

Abstract

Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great research interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNA(Gly) respectively, and reveal new aspects of the glycylation mechanism. We discover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunit fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases).

Keywords: Charcot-Marie-Tooth disease (CMT); aminoacyl tRNA synthetase; conformational change; crystal structure; enzyme mechanism.

FIGURE 1.

Overall structures of E71GFL and insertion 3. A, the GlyRS domain architecture without the N-terminal mitochondria localization signal. The catalytic domain (colored blue) contains three insertions (Ins1–3, colored magenta, green, and red, respectively) and three signature motifs (motifs 1–3, colored hot pink, yellow, and wheat, respectively). The anticodon binding domain (ACBD) is shown in cyan, and WHEP-TRS domain is shown in gray. The blue arrows indicate the positions of the two mutations in sequence, whereas the red arrows designate the start site of the two GlyRS constructs used in this work. B, ribbon representation of the closed conformation of the E71GFL apoenzyme. The color scheme is as in Fig. 1A. The disordered WHEP-TRS domain is modeled in and circled by the broken gray oval. The positions of CMT-causing residues Gly⁷¹ and other four residues falling into insertions 1 and 3 (Asp¹⁴⁶, Ser²¹¹, Asp⁵⁰⁰, and Cys¹⁵⁷) are indicated and labeled in the structure. C, structure comparison of the WT enzyme (yellow) and E71GFL (color scheme as in A). The resolved insertion 3 in E71GFL is noted by the red oval. D, the color-coded secondary structure of E71GFL. The α-helices, β-sheets, and β-hairpins are denoted as α, β, and η, respectively.

FIGURE 2.

Overall structures of the quaternary complex and conformational changes in insertion 3 on tRNA binding. A, the E71G/C157RSF-tRNA^Gly-AMPPNP complex in an open conformation. tRNA is in orange, whereas glycine and AMPPNP are in space-filling representation. The shape of the protein is labeled as parts of a hand. B, two orthogonal views of the dimer complex in ribbon rendition. The occupied monomer is colored as in Fig. 1A, whereas the other subunit is colored gray. C, the overlay of the structure of apo E71GFL and E71G/C157RSF-tRNA^Gly-AMPPNP complex. Although the AD domains superimpose well, both insertions show large displacements upon tRNA binding as indicated by the red arrows. The two insertions are circled. D, the structure superposition of E71G/C157RSF-tRNA^Gly-AMPPNP complex (Protein Data Bank code 4QEI), GlyRS-GlySA complex (Protein Data Bank code 2ZT8, purple), and E71GSF-tRNA^Gly-AMPPNP-glycine complex (Protein Data Bank code 4KR3, cyan). The AMPPNP moiety in all three structures superpose well.

FIGURE 3.

MD simulation of insertions 1 and 3 on a 20-ns scale. The vertical axis represents the RMSD of the insertions off their normal positions. The insertion 1 is colored in red, and the insertion 3 is colored in green. Insertion 1 undergoes larger conformational changes (RMSD 11.8 Å on average) than insertion 3 (RMSD 6.3 Å). The catalytic domain (black curve) serves as an internal control.

FIGURE 4.

The enzyme-tRNA interactions in the quaternary complex and the activity assays. A, interactions of insertion 3 and crossly bound tRNA^Gly, where tRNA is colored gray, and insertion 3 is colored red. The important residues that form contacts are shown in stick representation, and the elbow-contacting helix Lys⁴⁴⁷–Lys⁴⁵⁶ is colored yellow. The detailed enzyme-tRNA interactions in the quaternary complex. B, the contacts between the tRNA variable region and residues from the hinged loops in insertion 3. C, glycylation assays for mutants of residues involved in critical tRNA recognition. E71G/C157R represents the E71G/C157RSF double mutant, and all other mutants are in the short form (no WHEP-TRS domain). Two sets of data are shown, representing the measurements at 2- (blue) and 5-min time points (red) respectively. The activity of WT hGlyRS at the 5-min time point is regarded as 100%, and the readings at time 0 of the variants are used as blanks. The activity of each mutant is compared with WT in terms of percentages, and the error bars were calculated from two measurements. D, the shape complementation between the elbow region of tRNA and the α14 helix in insertion 3. Possible interactions are indicated by the red lines, and distances over 3.6 Å are indicated by the numbers. E, EMSA between GlyRS mutants and tRNA^Gly. ND, not determined. The K_d values are derived from the equation tRNA_bound/tRNA_total = [protein]/(K_d + [protein]) and given in the upper right table.

FIGURE 5.

PPi-exchange activity of WT and mutants of hGlyRS. The reactions were carried out using 100 nm enzymes. Formation of enzyme-bound aminoacyl-adenylate intermediate allows conversion of PPi into ATP. The reactions were quenched at different time points (0, 1, 2, 4, 8, and 16 min), separated on cellulose polyethyleneimine TLC plate, and visualized by a phosphorimaging plate. As shown in the top right corner, the insertion 3 point mutations or deletions did not greatly affect PPi exchange activities, when compared with the WT.

FIGURE 6.

Impacts of insertion 3 restraints on enzyme activities. A, secondary structure of insertion 3 in E71GFL. The major structural elements are labeled above the sequence, whereas residues of interests are designated by red dots below. B, the close-up view of insertion 3 in E71GFL and the structural elements are labeled. Several cysteine residues are shown, and their importance is tested as described below. Cys⁴⁶⁸ is located on a loop between α15 and α16, whereas Arg⁴²⁰ is on α13. C, activity tests of the cysteine mutants. O indicates oxidation with 1 mm oxidized glutathione 1 h at 4 °C; R indicates oxidation with 1 mm oxidized glutathione for 1 h at 4 °C followed by 10 mm DTT reduction for overnight at 4 °C. Two time points were used, and the data were processed as in C.

FIGURE 7.

Two crystal-packing patterns. A, the crystal-packing pattern in E71GFL crystal structure, which is mainly maintained by charged interactions. B, the crystal-packing pattern in E71G/C157RSF-tRNA^Gly-AMPPNP structure. Only the protein molecules are drawn for clarity. The red lines indicate possible interactions.