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The crystal structure of the archaeal leucyl-tRNA synthetase and tRNA (leucine) complex

Aminoacyl-tRNA synthetases (aaRSs) are ligases which exclusively attach a particular amino acid to the 3'-end of its set of cognate tRNAs isoacceptors. It is a two-step reaction : first, an activated intermediate called aminoacyl-adenylate is synthesized from the amino acid and ATP. Then, the activated amino acid is transferred to the 3’-end of the tRNA. It leads to the formation of the aminoacyl-tRNA. The fidelity of protein synthesis depends on the accuracy of this reaction .

Structure of leucyl-tRNA synthetase (LeuRS)Structure of leucyl-tRNA synthetase (LeuRS)

Leucyl-tRNA synthetase from the Archaeon Pyrococcus horikoshii is large (967 residues - 113kDa) and monomeric . LeuRS catalyses the esterification of tRNAs Leu with Leucine. There are two classes of aaRSs. Leucyl-tRNA synthetase (LeuRS) belongs to the class I and more precisely to the class Ia. The class I enzymes have the Rossmann-fold domain (parallel β-sheet and α-helices) and the two characteristic motifs, with the consensus sequences of His-Ile-Gly-His (HIGH) and Lys-Met-Ser-Lys-Ser (KMSKS). This family is divided into prokaryotic and eukaryal/archaeal groups but we will focus on the second group.

spinqualitylabelsall models Crystal Structure of Leucyl-tRNA Synthetase from the Archaeon Pyrococcus horikoshii resolution 2.05Å (PDB entry : 1wkb) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The structure can be divided into nine domains  :

1) The class I-characterizing : It contains the HIGH motif which is located at the beginning of the long α-helix found below the Rossmann-fold core β-sheet. Other the Rossmann fold domain, there are other fused and inserted domains:

2) The and 3) The consist of antiparallel β-sheets, but only the CP-core is fixed on the aminoacylation domain. There is a formed by the junction of the CP-core and the CP1 hairpin (Ala180 and His462 residues) which is essential for accommodating the tRNA .

4) The is located in the middle of the CP1 hairpin. It is the editing domain, that means that this region is responsible for the hydrolysis of the incorrectly synthesized products. Because LeuRS have a specific editing activity that hydrolyzes misactivated aminoacyl-adenylates (pre-transfer editing) and mischarged aminoacyl-tRNAs (post-transfer editing) or both when they have misformed with noncognate amino acids.The is necessary for post-transfer editing .

5) The is inserted between the second and third β-sheets of the CP core. It is composed of a pair of antiparallel α-helices and a connecting β-sheet. This domain is necessary for the amino acid activation and post-transfer editing .

6) The and 7) have been described in Archaeal LeuRs, inserted between the two helices in Rossmann-fold domain. They vary in size. LSD1 is a small domain which includes two antiparallel α-helices. The crystal structure of the apo enzyme suggests that LSD1 may have the potential to regulate the KMSKS loop opening and closing. This loop is highly dynamic and flexible during amino acid activation. LSD2 is a large domain (5 bundle α-helices) located on the opposite site of the enzyme relative to the aminoacylation active site .

8) The follows the Rossmann-fold domain. His β-α-α-β-α topology is characteristic. It contains the , which is located on the loop between the first β-sheet and the first α-helix of its topology .

9) The is found at the C-terminal end of the enzyme and consists of five long α-helices. It is essential for the tRNA binding throughout the tRNA Leu charging reaction.

Identity elements involved in complex formationIdentity elements involved in complex formation

spinqualitylabelsall models The crystal structure of Leucyl-tRNA synthetase and tRNA(leucine) complex(PDB entry 1wz2) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Most aaRSs interact with the tRNA anticodon loop in order to form aminoacyl-tRNA . But in the case of archaeal LeuRSs, none of them use this recognition mode. In fact, LeuRSs use two identity elements: the discriminator A73 and the long variable arm .

Recognition of the long variable armRecognition of the long variable arm

The first identity element in the complex is the recognition between the long variable arm of tRNA Leu and C-terminal residues of LeuRS . These residues recognize nucleotides localized at the tip of the arm but not at the stem region of the arm. This characteristic avoids interactions of tRNA Leu with SerRS and TyrRS, which also recognize tRNA with a long variable arm. It is not a direct interaction but a pocket is formed for a tip of the variable arm .

The C-terminal region of LeuRS is composed of β-sheet surrounded by α-helices and an additional region (a structure with α- α - α topology), specific to this enzyme. At the tip of the long variable arm of tRNA Leu, there are four nucleotides: G47a, U47b, A47c and G47d which formed a loop. Some of these nucleotides interact with C-terminal residues through Van der Waals interactions and hydrogen bonds. First, A47c and G47d which are turned outward of the loop interact with and . Then, A47c interacts specifically with : Ile849, Ile966 and Glu967. And G47d interacts with : Ile 849, Ile964, Asp845 and Pro962. Both A47c and G47d recognition by C-terminal region of the enzyme are important for recognition of the variable arm and a correct translation process .

Interactions with the discriminator, A73Interactions with the discriminator, A73

LeuRS employs the discriminator A73 as second identity element . A73 is localized in the tRNA 3’ region which has two conformations: the canonical ‘aminoacylation state’ and the ‘intermediate state’. Consequently, there is two recognition modes of A73, depending on complex structure.

Structural differences between the two complexesStructural differences between the two complexes

The first complex is observed when the CCA end localized at the 3' end of tRNA Leu is located in the aminoacylation active site; it is the aminoacylation complex.

The second structure, the intermediate complex, is observed when the CCA end is relocated toward the editing active site.

The translocation between these two states seems to be allowed thanks to the flexibility of the CCA end. It is able to go towards either the aminoacylation site either the editing site whereas the rest of the tRNA remains bound to the enzyme core. These two conformation states involved two modes of A73 recognition and also of C74, C75 and A73, residues of the 3’-terminal region .

Interactions with A73 and C74Interactions with A73 and C74

	The editing complex	The aminoacylation complex
Conformation mode	A73 and C74 stack together	A73 and C74 are perpendicular to each other
Recognitions mode A73	Hydrogen bond between N1 of A73 and NH of Asp504 in the main chain	Hydrogen bond between N6-H2 of A73 and CO of Lys 505 in the main chain Hydrogen bond between N1 of A73 and NH of Asp504
Recognition mode C74	Hydrogen bond between N4-H2 of C74 and CO of Lys505 in the main chain Hydrogen bond between N3 of C74 and NH of Ala507 in the main chain Hydrogen bond between 2’OH of C74 and COO- of glu527 in the side chain	Hydrogen bond between 2’OH of C74 and the positively charged Lys506 in the side chain Hydrogen bond between N3 of C74 and NH of Ala507 in the main chain

Interactions with C75 and A76Interactions with C75 and A76

The editing complex	The aminoacylation complex
The phosphate group between C74 and C75 interacts with the arg510 side chain	The phosphate group between C74 and C75 interacts with the arg510 side chain
The phosphate group between C75 and A76 interacts with the ser85 and ser528 side chains	The phosphate group between C75 and A76 do not interact with serine residues
Hydrogen bond between N6-H2 of A76 and Asn616 in the side chain	Hydrogen bond between N7 of A76 and Asn616 in the side chain
Hydrogen bond between 3’OH of A76 and His81 in the side chain

ContributorsContributors

Marion Vandeputte, Perrine Faiderbe