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). Aminoacyl-tRNA synthetases (aaRSs)2 play essential roles in mediating genetic information transfer from mRNAs to proteins and attach amino acids to their cognate tRNA molecules in a two-step reaction. In the first step of the reaction, the enzymes catalyze the condensation between ATP and the specific amino acid to generate an aminoacyl-adenylate intermediate. In the second, they transfer the activated amino acid to the acceptor stem of cognate tRNA to form the product aminoacyl-tRNA. The 20 aaRSs can be grouped into two distinct classes based of the conservation of primary sequences and quaternary structures (1-7). Glycyl-tRNA synthetase (GlyRS) is a class II enzyme and possesses three conserved signature motifs at the active site. However, different from other aaRSs, the quaternary structures of GlyRSs are not conserved phylogenetically. Two oligomeric forms have been discovered in nature: whereas eukaryotic and archaeal GlyRSs form ␣2 homodimers and belong to subclass IIa, their eubacterial counterparts form ␣2␤2 heterotetramers and belong to subclass . No significant sequence homology can be found between the two subtypes. In addition to the sequence and structure diversity, studies also show that GlyRSs only aminoacylate tRNA molecules within their own kingdoms, and crossspecies glycylation is rare. This phenomenon may be attributed to the distinct discriminator base at position 73. In eukaryotes, it is an adenosine that precedes the 3Ј-CCA end, whereas in prokaryotes, a uridine is present (9,11,14,15). Therefore, the tRNA recognition mode is an interesting problem, but it is poorly understood.The human GlyRS (hGlyRS) is a class IIa synthetase and forms a homodimer using motif 1. Motifs 2 and 3, on the other hand, are responsible for recognizing the substrates glycine and ATP. Additionally, hGlyRS features an N-terminal WHEP-TRS domain, as well as several insertion domains named insertions 1-3 (9, 16), most of which are flexible in structure (17). Recent studies have shown that aaRSs have developed functions other than aminoacyl...

Aminoacyl-tRNA synthetase evolution and sectoring of the genetic code

“…The closest structure identified using the Phyre2 server was human GlyRS-IIA (i.e. PDB 4KQE and 4QEI) [27,28]. Although human GlyRS-IIA lacks cysteine ligands for Zn binding, the fold of the homologous region of the protein is maintained, so a model of the conserved Pfu GlyRS-IIA Zn-binding domain was obtained.…”

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10.1080/21541264.2018.1467718-f0003Figure 3.A homology model (Supplementary File 1) of Pyrococcus furiosis GlyRS-IIA was constructed by homology threading to human GlyRS-IIA (PDB 4KQE). The homology model (powder blue), PDB 4KQE [28] (white) and related PDB 4QEI [27] (magenta) were overlaid. Although human ( Hs ) GlyRS-IIA lacks Zn binding, the shape of the loops is maintained.

10.1080/21541264.2018.1467718-f0004Figure 4.A structurally conserved Zn-binding motif among class I and class II aaRS enzymes.

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“…The homology model (powder blue), PDB 4KQE [28] (white) and related PDB 4QEI [27] (magenta) were overlaid. Although human ( Hs ) GlyRS-IIA lacks Zn binding, the shape of the loops is maintained.…”

“…Pfu GlyRS-IIA was modeled to human GlyRS-IIA (PDB 4KQE) [28] using the program Phyre2 [45,46]. Atomic coordinates were refined using the YASARA energy minimization server (http://www.yasara.org/minimizationserver.htm).…”

Drugging tRNA aminoacylation

“…Furthermore, a co-crystallized inactive complex of E. coli AspRS and yeast tRNA Asp supports a distinct tRNA-initiated control mechanism that initiates loop movement in the AspRS catalytic domain, opening the acceptor stem site of AspRS [96]. A serendipitous high-resolution structure of human GlyRS demonstrated two distinct conformational states of GlyRS recognition during tRNA complexation within a single crystal lattice, supporting conformational change in GlyRS loop regions to allow tRNA acceptor stem entry for the first step of aminoacylation [111]. A high degree of flexibility in this region during both tRNA binding and aminoacylation was further supported by limited molecular dynamics studies [111].…”