Human Tryptophanyl-tRNA Synthetase Recognition and Specificity

Christiana Binkley '17 and Grace Riley '18

Contents:

I. Introduction

The field of molecular biology revolves around the central dogma that DNA is transcribed to form RNA which is translated to synthesize a protein. Many macromolecules are necessary to carry out the numerous processes that lead to a functional protein. The enzyme, aminoacyl tRNA synthetase (aaRS) is imperative in protein production, as it must correctly charge the tRNA with its corresponding amino acid.1 There are two classes of aaRTs. Class I synthetases share a similar folding motif in the catalytic domain and they perform aminoacylation with the 2'-OH from the tRNA acceptor arm, interacting in the minor groove. Class II synthetases have a different, distinct folding motif as well. Aminoacylation is performed on the 3'-OH group, and the aaRS interacts with the tRNA in the major groove. Here we model the complex of human , , and tryptophan, shown below. The protein exists biologically as a dimer, so the complex includes two tRNAs, two tryptophans, and the homodimer. This synthetase is unique in that it exhibits both class I and class II behaviors. The tRNA binds in a conformation that is characteristic of a class I binding mechanism. However, the synthetase receives the tRNA in the major groove of the acceptor arm, indicative of class II complexes.2 Structure of tryptophan:3

II. General Structure

The human tryptophanal-tRNA synthetase is a homodimer composed of three domains: N-terminal fragment, catalytic domain, and the C-terminal domain . The N-terminal fragment interacts with the tRNA acceptor arm. Unfortunately, the 5'-CCA-3' acceptor arm did not crystallize, so it is not depicted here. The catalytic domain has a Rossmann Fold motif--a highly conserved polypeptide motif characterized by the alternation of beta sheets and alpha helices--and a connective polypeptide 1 insertion . The CP1, which is composed of the α5 and α6 helices, is important in the dimer interface interactions, and it helps form the substrate-binding pocket. Helices in the C-terminal domain aid in formation of the . Two tRNA's and two tryptophans were crystallized in the complex, one of each associated with a unit of the dimer.2

One is located in the of each synthetase unit. The tryptophan forms hydrogen bonds and pi-pi stacking interactions with residues in the pocket, depicted below. The hydrogen bond with Gln194 and pi-pi interactions with Tyr159 are most important in recognizing and binding tryptophan.2

III. tRNAtrp

The acceptor arm of the tRNA interacts with three helices of the synthetase (helices α1', α6, and α9). These helices help guide the uncharged tRNA acceptor arm into the the catalytic pocket where aminoacylation will occur. In this model, the conserved 5'-CCA-3' end of the acceptor arm was not crystallized due to its flexible nature. At the 5' end of the CCA lies the , A73. This base participates in hydrogen bonding with residues in the synthetase, allowing the synthetase to exercise selectivity for only tRNAs with A73. Research suggests that play the largest role in selecting for the A73 discriminator base. For example, when Asp99 was mutated to either alanine or valine, which are both hydrophobic and smaller than aspartate, a significant reduction in the synthetase's aminoacylation activity was observed. The size and polarity of these residues is crucial for facilitating interactions with the tRNAtrp discriminator base, and hence permits proper synthetase activity.2

Further selectivity for the correct tRNA can be observed in the of the tRNA. The 5'-CCA-3' anticodon (coincidentally the same sequence as the 3' end of the acceptor arm) interacts with helices α10, α11, and α14 of the synthetase, comprising the . The anticodon base A36 exhibits minimal specific interactions with the anticodon pocket, compared to C35 and C34 which exhibit more hydrogen bonding with the synthetase.2

IV. Aminoacylation Reaction

Aminoacylation is the process in which tRNA is charged with an amino acid. The reaction occurs in two steps. First, the tryptophan is activated by ATP to form aminoacyl adenylate and pyrophosphate. Then, the 2'-OH on the terminal ribose of the tRNA acceptor arm nucleophilically attacks the carbonyl in the amino acid backbone, displacing the AMP.5 Click for mechanism.6 This reaction occurs in the of the tryptophanal tRNA synthetase. The synthetase guides the to the catalytic pocket and holds the tRNA in the correct orientation so the reaction can occur rapidly and readily.5

Before aminoacylation occurs, the synthetase undergoes a conformational change that induces activation of the tryptophan and promotes entrance of the tRNA acceptor arm into the catalytic domain. Conformational changes have also been observed on the tRNA acceptor arm during aminoacylation. The structure presented here is of the synthetase before tryptophan activation and tRNA charging. One structure within the synthase that facilitates this reaction is the beta hairpin (not crystallized), located just above the tryptophan binding pocket. It is thought that this hairpin is necessary for the aminoacylation reaction, after experiments using synthetase mutants lacking the beta hairpin failed to perform aminoacylation. Pro87 and Trp88 (not crystallized) are residues in the beta-hairpin that are highly conserved within eukaryotic and archael tryptophan aaRSs, and they perform hydrogen bonding with . The latter two residues also aid in tryptophan activation. Overall, the aminoacylation reaction involves a variety of protein-tRNA interactions and conformational changes, and is necessary for translation and cell survival.2

V. References

(1) Watson, James D., Tania A. Baker, Stephen P. Bell, Alexander Gann, Michael Levine, and Richard Losick. Molecular Biology of the Gene.Glenview, IL. Pearson, Cold Spring Harbor Laboratory P. 2014. Print.

(2) Shen, Ning, et al. "Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity ." Nucleic Acids Research 34.11 (2006): 3246-3258.

(3) Tryptophan Chemical Structure. N.d. Amino Acid Structures. About Education. By Todd Helmenstine. Web. 07 Dec. 2015.

(4) Phil. "Topology in 2D and 3D- The Rossmann Fold." Web log post. Protein Portraits. OSU Honors College, 16 Apr. 2012. Web. 3 Dec. 2015.

(5) Ibba, Michael, and Dieter Söll. "Aminoacyl-tRNA synthesis." Annual Review of Biochemistry 69.1 (2000): 617-650.

(6) Li, Rongzhong, et al. "Md simulations of tRNA and aminoacyl-tRNA synthetases: Dynamics, folding, binding, and allostery." International journal of molecular sciences 16.7 (2015): 15872-15902.

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