Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase

Abstract

Leucyl-tRNA synthetase (LeuRS) produces error free leucyl-tRNALeu by coordinating translocation of the 3′ end of (mis-)charged tRNAs from its synthetic site to a separate proof-reading site for editing. Here we report co-crystal structures of the Escherichia coli LeuRS-tRNALeu complex in the aminoacylation or editing conformations and show that translocation involves correlated rotations of four flexibly linked LeuRS domains. This pivots the tRNA to guide the charged tRNA 3′ end from the closed aminoacylation state to the editing site. The editing domain unexpectedly stabilizes the tRNA during aminoacylation while a large rotation of the leucine-specific domain positions the conserved KMSKS loop to bind the 3′ end of the tRNA, promoting catalysis. Our results give new insight into the structural dynamics of a molecular machine that is essential for accurate protein synthesis.

LARS2 Variants Associated with Hydrops, Lactic Acidosis, Sideroblastic Anemia, and Multisystem Failure

“…This view is supported by the fact that in E. coli LARS, a unique mutation in CP1 affects leucylation activity (Du and Wang 2003). In addition, structural data show that CP1 is directly involved in positioning tRNA Leu on E. coli LARS for efficient charging (Palencia et al 2012). However, because the editing-inactive CP1 domain of yeast LARS2 is required for splicing of group I introns (Sarkar et al 2012), it is possible that an unknown alternative function could be altered in the CP1 domain of human LARS2 p.Ala430Val.…”

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

“…In the Leu-AMS functional aminoacylation state, this ribose is in a C2 0 -exo conformation, allowing the 2 0 -OH to point towards the carbonyl-carbon of Leu-AMS (Fig. 5b,c) as would be expected prior to the transfer step in the aminoacylation reaction 21 . In the TM84 complex, the Ade76 ribose is in an unexpected C2 0 -endo conformation, resulting in the 2 0 -OH pointing away from the carbonyl-carbon and instead forming a hydrogen bond to a nearby water molecule that in turn hydrogen bonds to Asp80 and Ser496 (Fig.…”

“…Surprisingly, we now show that TM84 is different from Leu-AMP analogues, as it employs a unique tRNAdependent inhibition mechanism in which the 3 0 -end of tRNA Leu is required to form an 'aminoacylation-like' conformation that stabilizes the binding of the inhibitor in a ternary inhibition complex. Our structure, together with a recently published structure of an Escherichia coli LeuRS Á tRNA Leu Á Leu-AMP analogue 21 , reveal exciting new insights into the recognition of tRNA Leu by LeuRS during aminoacyl transfer, and molecular details of how the enzyme catalyses the second stage of the aminoacylation reaction.…”

“…To understand how TM84 binds to LeuRS and gain structural insights into the role of tRNA Leu in the unusual LeuRS inhibitory properties of TM84, we crystallized LeuRS in complex with tRNA Leu and TM84. We used recombinant E. coli LeuRS (LeuRS Ec ) and in vitro transcribed E. coli tRNA Leu (UAA) isoacceptor substrates, which have recently been shown to produce high diffraction-quality crystals 21 (Fig. 4a).…”

“…To identify key interactions in the LeuRS ternary complex that may be responsible for the tRNA-dependence of TM84 binding, we made comparisons with a structure of LeuRS Ec Á tRNA Leu Á Leu-AMS captured in the functional aminoacylation conformation 21 . Leu-AMS, unlike TM84, does not show tRNAdependent binding to LeuRS and as a consequence the tRNA could occupy more than one conformation on the enzyme in solution, an observation that is in accordance with our fluorescence data (Fig.…”