Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.Aminoacyl-tRNA synthetases (AARSs)⁴ are considered to be among the earliest proteins to have emerged during evolution. As a family of typically 20 members (one for each amino acid), AARSs catalyze the first step of protein synthesis by linking each amino acid onto the 3′-end of its cognate tRNA harboring the trinucleotide anticodon. Through evolution, the role of AARSs has also been broadened with expanded functions (reviewed in Refs. 1 and 2). These expanded functions often involve direct interaction partners. For example, human tyrosyl-tRNA synthetase interacts with chemokine receptor CXCR1 to induce cell migration (3); human glutaminyl-tRNA synthetase interacts with ASK1 to regulate apoptosis (4); human tryptophanyl-tRNA synthetase interacts with VE-cadherin to inhibit angiogenesis (5); human lysyl-tRNA synthetase interacts with the Gag protein of human immunodeficiency virus to facilitate viral assembly (6); and human glutamyl-prolyl-tRNA synthetase interacts with L13a and glyceraldehyde-3-phosphate dehydrogenase to form the GAIT complex for translational silencing to regulate inflammation (7). However, functional expansion also can be achieved indirectly via reaction products of AARSs. As examples, Lys-tRNA^Lys and Ala-tRNA^Ala are used to aminoacylate cytoplasmic membrane phosphatidylglycerol of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, to enhance drug resistance in these microorganisms (8).In addition to tRNA aminoacylation, the majority of AARSs have the capacity to catalyze a side reaction to form diadenosine oligophosphates (ApnA) in the absence of cognate tRNA (9). These reactions of AARS are the most well known sources of ApnA in vivo (10). ApnA are made up of two adenosine moieties linked at the 5′-end of the ribose by a chain of two to six phosphates. In the 4 decades following the discovery of these molecules by Zamecnik et al. (10), ApnA have been linked to highly diverse physiological effects in prokaryotic and eukaryotic cells, including various types of mammalian cells and tissues, and to assorted functions associated with the nucleus, membrane receptors, and activities in the cytoplasm (reviewed in Refs. 11 and 12). The concentrations of ApnA molecules in vivo respond to numerous factors, including cell proliferation status, glucose level, heat shock, oxidative stress, and interferon stimulation. They have emerged as extracellular and intracellular signaling molecules (as pleiotropically acting “alarmones” (13) and second messengers (14)) implicated in the maintenance and regulation of vital cellular functions.The aminoacylation reaction proceeds in two steps. First, the amino acid is activated by condensation with ATP to form aminoacyl-AMP, the enzyme-bound intermediate. The aminoacyl moiety is then transferred to the 3′-end of the cognate tRNA. When tRNA is absent, the enzyme-bound aminoacyl-AMP can be attacked by the γ-phosphate of a second ATP molecule to form diadenosine tetraphosphate (Ap4A), the most common diadenosine oligophosphate produced by a tRNA synthetase (see Fig. 1A). The presence of tRNA in most cases inhibits Ap4A synthesis (11). Therefore, a subgroup of tRNA synthetases that requires tRNA as cofactor for synthesis of aminoacyl-AMP is not capable of producing Ap4A. This group includes tRNA synthetases that are specific for arginine, glutamine, and glutamic acid and an unusual class I lysyl-tRNA synthetase (LysRS).Open in a separate windowFIGURE 1.Amino acid-independent synthesis of Ap4A by human GlyRS. A, conventional mechanism for synthesis of Ap4A by a tRNA synthetase. The first step of the reaction involves the generation of an enzyme-bound aminoacyl-AMP (aa-AMP), which is then attacked either by cognate tRNA to form aminoacyl-tRNA or by ATP to form Ap4A. B, mechanism used by human GlyRS to produce Ap4A by direct condensation of two ATPs. C–E, synthesis of Ap4A by GlyRS, LysRS, and TyrRS, respectively, in the presence (●) and absence (○) of cognate amino acid. Amounts of Ap4A produced were quantitated from the TLC sheets and are plotted on the right. GlyRS activity was measured in triplicate, and the plotted values reflect the mean ± S.E.Although the amino acid recycles, the above mechanism requires the presence of the amino acid for the production of Ap4A via the aminoacyl-AMP intermediate. Using biochemical analyses and determinations of co-crystal structures, we demonstrate in this work that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs in the absence of glycine. Thus, the mechanism for GlyRS to synthesize Ap4A is decoupled from aminoacylation. Furthermore, GlyRS is likely to be the only synthetase that produces Ap4A by this mechanism. Our results raise the possibility that GlyRS plays a special role in Ap4A homeostasis.